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WO2019066549A2 - Manipulation de gène pour le traitement d'un trouble de dysfonctionnement rétinien - Google Patents

Manipulation de gène pour le traitement d'un trouble de dysfonctionnement rétinien Download PDF

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Publication number
WO2019066549A2
WO2019066549A2 PCT/KR2018/011522 KR2018011522W WO2019066549A2 WO 2019066549 A2 WO2019066549 A2 WO 2019066549A2 KR 2018011522 W KR2018011522 W KR 2018011522W WO 2019066549 A2 WO2019066549 A2 WO 2019066549A2
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sequence
nucleic acid
gene
domain
retinal function
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Korean (ko)
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WO2019066549A3 (fr
Inventor
이정민
송동우
김운기
김정훈
조동현
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Toolgen Inc
SNU R&DB Foundation
Seoul National University Hospital
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Toolgen Inc
Seoul National University R&DB Foundation
Seoul National University Hospital
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Priority to US16/650,993 priority Critical patent/US11845951B2/en
Priority to EP18862381.3A priority patent/EP3690045A2/fr
Publication of WO2019066549A2 publication Critical patent/WO2019066549A2/fr
Publication of WO2019066549A3 publication Critical patent/WO2019066549A3/fr
Anticipated expiration legal-status Critical
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N2750/14011—Parvoviridae
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Definitions

  • the present invention relates to a gene manipulating composition for treating or ameliorating a retinal dysfunction disorder or a method using the same. More specifically, a gene manipulating composition comprising a guide nucleic acid capable of targeting a retinal function-forming gene and a method for treating or improving a disease caused by retinal dysfunction by artificially manipulating and / or calibrating a retinal function- .
  • LCA which causes blindness in newborn babies, does not have the right treatment yet. It is caused by genetic variation of over 20 different genes.
  • LCA from Spark Therapeutics, etc. Of RPE65, but there is a disadvantage that the treatment effect is obtained only while AAV is present. Therefore, for longer-term therapeutic effects, a method of correcting mutated mutations is needed.
  • the present invention provides a guide nucleic acid that targets a retinal function-forming gene to artificially manipulate a retinal function-forming gene.
  • the present invention provides a gene manipulating composition for artificially manipulating a retinal function-forming gene.
  • a method for artificially manipulating a retinal function-forming gene is provided.
  • a method for treating a retinal dysfunction disease using a gene-acting composition As one embodiment of the present invention, there is provided a method for treating a retinal dysfunction disease using a gene-acting composition.
  • the present invention relates to a gene-acting composition for treating a retinal dysfunctional disease. More particularly, the present invention relates to a gene manipulating composition capable of artificially manipulating a retinal function-forming gene for treating a retinal dysfunction disease and a method using the same.
  • the present invention provides a guide nucleic acid capable of targeting a retinal function-forming gene.
  • the retinal function-forming gene may be the RPE65 gene.
  • the guide nucleic acid can target the target sequence of the retinal function forming gene.
  • the guide nucleic acid may include a guide domain capable of targeting a target sequence of a retinal function type gene.
  • the guide domain may include a nucleotide sequence capable of forming a complementary bond with a guide nucleic acid binding sequence in a target sequence of a retinal function-forming gene.
  • the guide domain may form a complementary bond with a guide nucleic acid binding sequence in a target sequence of a retinal function forming gene.
  • the complementary combination may include 0 to 5 mismatching combinations.
  • the guide nucleic acid may comprise one or more domains selected from the group consisting of a first complementary domain, a connecting domain, a second complementary domain, a proximal domain and a tail domain.
  • the target sequence may comprise or be located in close proximity to a mutant sequence of a retinal function forming gene.
  • the mutant sequence of the retinal function-forming gene is a part of a retinal function-forming gene in which at least one nucleotide has been deleted, inserted or substituted, as compared with a nucleic acid sequence of a wildtype retinal function- Nucleic acid sequence.
  • the mutation sequence of the retinal function-forming gene may be a partial nucleic acid sequence of a retinal function-forming gene having at least one other codon sequence as compared to a nucleic acid sequence of a wildtype retinal function-forming gene.
  • the mutant sequence of the retinal function-forming gene may be located in at least one region selected from the group consisting of an exon region and an intron region of a retinal function-forming gene.
  • the mutant sequence of the retinal function-forming gene may be located in the exon region of the RPE65 gene.
  • the target sequence may be located in at least one region selected from the group consisting of a promoter region, an exon region, an intron region, and an enhancer region of a retinal function-forming gene.
  • the target sequence may be a sequence of 10-25 nucleotides contiguous to the 5 'end and / or the 3' end of a proto-spacer-adjacent Motif (PAM) sequence in the nucleic acid sequence of the retinal function forming gene.
  • PAM proto-spacer-adjacent Motif
  • the target sequence may be a sequence of 10-25 consecutive contiguous contiguous 5 ' and / or 3 ' ends of a proto-spacer-adjacent Motif (PAM) sequence in the nucleic acid sequence of a retinal function- forming gene comprising a mutated region of a retinal function- Lt; / RTI > nucleotide sequence.
  • PAM proto-spacer-adjacent Motif
  • the target sequence may be at least one selected from SEQ ID NOS: 1-69.
  • the present invention provides a gene manipulation composition for artificially manipulating a retinal function-forming gene.
  • the genetic engineering composition may comprise:
  • a guide nucleic acid capable of targeting a target sequence of a retinal function forming gene or a nucleic acid sequence encoding the same
  • Cas9 protein derived from Streptococcus pyogenes Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Staphylococcus aureus (Staphylococcus aureus) a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein, or a nucleic acid sequence encoding the same.
  • the retinal function-forming gene may be an RPE65 gene.
  • the target sequence may comprise or be located in close proximity to a mutant sequence of a retinal function forming gene.
  • the mutant sequence of the retinal function-forming gene may have a retinal function including a deletion, insertion or substitution of one or more nucleotides in comparison with a nucleic acid sequence of a wildtype retinal function- May be part of the nucleic acid sequence of the gene.
  • mutant sequence of the retinal function-forming gene may be a partial nucleic acid sequence of a retinal function-forming gene comprising at least one other codon sequence as compared to the nucleic acid sequence of a wildtype retinal function-forming gene.
  • the mutant sequence of the retinal function-forming gene may be located within the exon region of the retinal function-forming gene.
  • the guide nucleic acid and the editor protein may form a guide nucleic acid-editor protein complex.
  • the guide nucleic acid-editor protein complex may be formed by interacting some nucleic acids of the guide nucleic acid and some amino acids of the editor protein.
  • the guide nucleic acid and the editor protein may each be present as one or more vectors in the form of nucleic acid sequences.
  • the vector may be a plasmid.
  • the vector may be one or more viral vectors selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus.
  • retrovirus lentivirus
  • adenovirus lentivirus
  • AAV adeno-associated virus
  • vaccinia virus poxvirus
  • herpes simplex virus herpes simplex virus.
  • the genetic engineering composition may comprise:
  • a guide nucleic acid capable of targeting a target sequence of a retinal function forming gene or a nucleic acid sequence encoding the same;
  • Cas9 protein derived from Streptococcus pyogenes Cas9 protein derived from Campylobacter jejuni
  • Cas9 protein derived from Streptococcus thermophilus Cas9 protein derived from Streptococcus thermophilus
  • Staphylococcus aureus Staphylococcus aureus
  • a cas9 protein derived from aureus a Cas9 protein derived from Neisseria meningitidis
  • Cpf1 protein or a nucleic acid sequence encoding the same a nucleic acid sequence encoding the same.
  • a donor comprising a nucleic acid sequence desired to be inserted or a nucleic acid sequence encoding the same.
  • the retinal function-forming gene may be an RPE65 gene.
  • the target sequence may comprise or be located in close proximity to a mutant sequence of a retinal function forming gene.
  • the mutant sequence of the retinal function-forming gene may have a retinal function including a deletion, insertion or substitution of one or more nucleotides in comparison with a nucleic acid sequence of a wildtype retinal function- May be part of the nucleic acid sequence of the gene.
  • mutant sequence of the retinal function-forming gene may be a partial nucleic acid sequence of a retinal function-forming gene comprising at least one other codon sequence as compared to the nucleic acid sequence of a wildtype retinal function-forming gene.
  • the guide nucleic acid and the editor protein may form a guide nucleic acid-editor protein complex.
  • the guide nucleic acid-editor protein complex may be formed by interacting some nucleic acids of the guide nucleic acid and some amino acids of the editor protein.
  • the nucleic acid sequence desired to be inserted may be a partial nucleic acid sequence of a retinal function-forming gene.
  • the nucleic acid sequence desired for insertion may be a normal nucleic acid sequence for correcting the mutation sequence of the retinal function-forming gene.
  • the donor may comprise a nucleotide sequence that is each homologous to a nucleotide sequence upstream and / or downstream of the truncated target sequence.
  • the nucleotide sequence having the homology may be a nucleotide sequence having at least 50% or more homology.
  • the guide nucleic acid, the editor protein, and the donor may each be present in one or more vectors in the form of nucleic acid sequences.
  • the vector may be a plasmid.
  • the vector may be one or more viral vectors selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus.
  • retrovirus lentivirus
  • adenovirus lentivirus
  • AAV adeno-associated virus
  • vaccinia virus poxvirus
  • herpes simplex virus herpes simplex virus.
  • the present invention provides a method for treating a retinal dysfunction disease.
  • a method of treating a retinal dysfunction disorder may comprise administering a gene therapy composition to a subject to be treated.
  • the gene manipulating composition may comprise:
  • a guide nucleic acid capable of targeting a target sequence of a retinal function forming gene or a nucleic acid sequence encoding the same
  • Cas9 protein derived from Streptococcus pyogenes Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Staphylococcus aureus (Staphylococcus aureus) a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein, or a nucleic acid sequence encoding the same.
  • the retinal function-forming gene may be an RPE65 gene.
  • the target sequence may comprise or be located in close proximity to a mutant sequence of a retinal function forming gene.
  • the mutant sequence of the retinal function-forming gene may have a retinal function including a deletion, insertion or substitution of one or more nucleotides in comparison with a nucleic acid sequence of a wildtype retinal function- May be part of the nucleic acid sequence of the gene.
  • mutant sequence of the retinal function-forming gene may be a partial nucleic acid sequence of a retinal function-forming gene comprising at least one other codon sequence as compared to the nucleic acid sequence of a wildtype retinal function-forming gene.
  • the gene manipulating composition may optionally further comprise a donor or nucleic acid sequence encoding it.
  • the retinal dysfunction disorder may be selected from the group consisting of Leber congenital darkness, retinitis pigmentosa, Starkart disease, retinopathy, color blindness, choroidal atrophy, macular degeneration, myopia, choroidal vasculopathy, central serous chorioretinopathy , Macular hole, macular dystrophy, diabetic retinopathy, retinal vein occlusion, hypertensive retinopathy, retinal aortic aneurysm, ocular ischemic syndrome, prematurity retinopathy, acute retinal necrosis, cytomegalovirus retinitis, toxoplasmic retinal choroiditis, Retinitis, retinal detachment, or retinoblastoma.
  • the retinal dysfunction disorder may be Leber congenital darkness or retinitis pigmentosa.
  • the subject to be treated may be a mammal including humans, monkeys, mice, and rats.
  • the administration can be performed by injection, transfusion, implantation or transplantation.
  • the administration can be carried out by a selected route of administration, subretinal, intraocularly, intravitreally, intramuscularly or intravenously.
  • the present invention can treat a retinal dysfunction disorder through a gene-acting composition. More specifically, a gene manipulating composition containing a guide nucleic acid targeting a retinal function-forming gene is used to artificially manipulate and / or calibrate a retinal function-forming gene so that the retinal function-forming gene can function normally or normally, Dysfunction can be improved or remedied.
  • Figure 1 is a schematic diagram of the wild-type Rpe65 gene.
  • the sequence corresponding to the early termination codon of rd12 mouse is indicated by an underline, the backward arrow ( ⁇ ) represents TS1 sgRNA target sequence, and the forward arrow ( ⁇ ) represents TS4 sgRNA target sequence , And the protospacer adjacent motif (PAM) was represented by a square box.
  • PAM protospacer adjacent motif
  • FIG. 2 is an image showing TS1 sgRNA and TS4 sgRNA-induced mutations in C2C12 cells detected by the T7E1 assay.
  • the control (Ctrl) was treated with only Cas9 and the DNA fragment cleaved by T7E1 was indicated with an arrow.
  • the ratio of SpCas9 to Rpe65-donor is shown in parentheses. Error bars indicate SEM, and were marked as *** P ⁇ 0.001 by Student's t test for one-way ANOVA and TS1 sgRNA with post-hoc Bonferroni's test for TS4 sgRNA.
  • FIG. 5 shows the average value of the number of deep sequencing leads in different categories.
  • FIG. 6 is an image showing a nucleotide sequence representing the Rpe65 donor template and a change induced by the TS4 sgRNA, wherein the inverted triangle represents the DSB position induced by the TS4 sgRNA and the sequence corresponding to the early termination codon of the rd12 mouse Are indicated by an underline (straight line), and PAM is indicated by a square box (dotted line).
  • the donor's synonymous mutations are indicated by underlining (wave)
  • Figure 7 is a schematic illustration of a dual AAV strategy for CRISPR / Cas9 mediated therapy correction of nonsense mutations of Rpe65 in rd12 mice, wherein the early termination codon (underlined) of Rpe65 is generated by a C? T mutation in exon 3,
  • the AAV vector used the U6 promoter and the EFS promoter for the expression of SpCas9 to express both the sgRNA and the donor template (the synonymous mutation is indicated by the underscore (wave)).
  • Therapeutic gene correction can be derived from HDR-mediated precision correction (HDR) or in-frame NHEJ (Inf-NHEJ).
  • Figure 8 shows the results of targeted deep sequencing in retina (a) and RPE (b) of rd12 mice at 4 weeks after high dose (total 2x10 11 vg / eye) and low dose (total 2x10 10 vg /
  • high dose total 2x10 11 vg / eye
  • low dose total 2x10 10 vg /
  • the ratio of SpCas9 to TS4 rd12 sgRNA- Rpe65 -donor is shown in parentheses.
  • 9 is a graph showing the correlation between the number of AAV copies per diploid cell and the indelible frequency.
  • FIG. 10 is a graph showing the frequency of therapeutic HDR at C? T mutation positions in the retina and RPE of rd12 mice at 4 weeks after injection.
  • FIG. 11 is a graph showing the ratio of indelible frequency to HDR.
  • FIG. 12 is a graph (a) showing the average value of the number of deep sequencing leads including insertion, deletion, or HDR in the RPE treated with AAV and a graph (b) showing the average percentage value of in-frame deletion, 1-codon deletion, or HDR )
  • Rpe65mut / mut or others refers to an untreated control, Ins insert, Del deletion, Inf-Del means in-frame deletion.
  • the inverted triangle ( ⁇ ) indicates the position of DSB induced by TS4 sgRNA
  • the sequence corresponding to the early termination codon of rd12 mouse is indicated by an underline (dotted line)
  • PAM is indicated by a square box.
  • the donor's synonymous mutations are indicated by underscores (undulations), and the nucleotide sequences containing the early termination codon and their corresponding amino acid sequences are indicated by underlines (straight lines).
  • Scale bar 20 ⁇ m. * P <0.05; ** P ⁇ 0.01 by Student's t test.
  • Scale bar 20 ⁇ m. * P <0.05; ** P ⁇ 0.01 by Student's t test.
  • Figure 17 shows the H & E image (a) of the retinal tissue of rd12 mice with or without SRT injection at 1: 1 ratio of SpCas9 and TS4 rd12 sgRNA- Rpe65 -donor at 7 months after injection, Rd12-AAV: SpCas9 and TS4 rd12 sgRNA- Rpe65 -donor at a ratio of 1: 1 (GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; rd12: untreated rd12 mouse; Rd12 mice injected under the retina and treated). Scale bar, 20 ⁇ m. * P <0.05; ** P ⁇ 0.01 by Student's t test.
  • Figure 18 is an image showing the expression of Rpe65 and HA in the RPE layer of rd12 mice (rd12: untreated rd12 mice; rd12-AAV: SpCas9 and TS4 rd12 sgRNA- Rpe65 -donor in a 1: Lt; / RTI > mouse). Scale bar, 20 ⁇ m.
  • FIG. 19 is a graph (a) showing the frequency of Indel at the human RPE65 target site of HEK293 cell line, a graph (b) showing the percentage value of in-frame and out-of-frame indel, Respectively.
  • the indelible frequency in the human target site (Ex3-2) corresponding to the TS4 target site is indicated by an arrow.
  • Figure 19 also shows a graph (c) with percentage values of in-frame and out-of-frame indels at 16 RPE65 target sites.
  • FIG. 20 shows a comparison between the human RPE65 and mouse Rpe65 genes and their amino acid sequences.
  • the sequences targeted by human (Ex3-2) and mouse (TS4) sgRNAs are shown underlined and PAM sequences are shown in square boxes.
  • Figure 21 is a graph showing the results of targeted deep sequencing and a list of potential off-target positions for the 33 homologous sites differing from the TS4rd12 target site and up to three nucleotides in the AAV-treated retina, The nucleotides are represented by lower case alphabet and the PAM sequence is indicated by a square box.
  • Figure 22 shows a representative H & E image of the RPE layer of rd12 mice at 7 months of untreated or treated SpAc9 and dual AAV systems encoding donor templates. Scale bar, 20 ⁇ m.
  • One aspect of the teachings disclosed herein relates to a guide nucleic acid.
  • Guided nucleic acid &quot refers to a nucleotide sequence that recognizes a target nucleic acid, gene or chromosome, and is capable of interacting with an editor protein.
  • the guide nucleic acid can be complementary to a target nucleic acid, a gene or a nucleotide sequence within a chromosome.
  • some nucleotide sequences of the guide nucleic acid may interact with some amino acids of the editor protein to form a guide nucleic acid-editor protein complex.
  • the guide nucleic acid may function to induce the guide nucleic acid-editor protein complex to be located in the target region of the target nucleic acid, gene or chromosome.
  • the guide nucleic acid may be in the form of a DNA, RNA or DNA / RNA blend, and may have 5 to 150 nucleic acid sequences.
  • the guide nucleic acid may be a single contiguous nucleic acid sequence.
  • one contiguous nucleic acid sequence may be (N) m , where N is A, T, C or G, or A, U, C or G, and m is an integer from 1 to 150 .
  • the guide nucleic acid may have two or more consecutive nucleic acid sequences.
  • two or more consecutive nucleic acid sequences may be (N) m and (N) o , wherein N is A, T, C or G, or A, U, C or G, Means an integer of 1 to 150, and m and o may be the same or different from each other.
  • the guide nucleic acid may comprise one or more domains.
  • the domain may be, but is not limited to, a functional domain such as a guide domain, a first complementary domain, a connecting domain, a second complementary domain, a proximal domain, or a tail domain.
  • a functional domain such as a guide domain, a first complementary domain, a connecting domain, a second complementary domain, a proximal domain, or a tail domain.
  • one guide nucleic acid may have two or more functional domains.
  • the two or more functional domains may be different from each other.
  • one guide nucleic acid may have a guide domain and a first complementary domain
  • one guide nucleic acid may have a second complementary domain, a proximal domain and a tail domain
  • another guide nucleic acid can have a guiding domain, a first complementary domain, a connecting domain, a second complementary domain, a proximal domain, and a tail domain.
  • two or more functional domains contained in one guide nucleic acid may be identical to each other.
  • one guide nucleic acid can have two or more proximal domains, and in another, one guide nucleic acid can have two or more tail domains.
  • the expression that the functional domains included in one guide nucleic acid are the same domain does not mean that the sequences of the two functional domains are the same, and if they function identically even if the sequences are different, they can be said to be the same domain .
  • a "guiding domain" is a domain capable of binding complementary to a partial sequence of a target gene or a double strand of a nucleic acid, and serves for specific interaction with a target gene or nucleic acid.
  • the guiding domain can function to induce a guide nucleic acid-editor protein complex to a position having a specific nucleotide sequence of the target gene or nucleic acid.
  • the guiding domain may be from 10 to 35 nucleotide sequences.
  • the guiding domain may be from 10 to 35 nucleotides, from 15 to 35 nucleotides, from 20 to 35 nucleotides, from 25 to 35 nucleotides, or from 30 to 35 nucleotides.
  • the guiding domain may be from 10 to 15 nucleotides, from 15 to 20 nucleotides, from 20 to 25 nucleotides, from 25 to 30 nucleotides, or from 30 to 35 nucleotides.
  • the guide domain may comprise a guide sequence.
  • a "guide sequence” is a nucleotide sequence complementary to a partial sequence of a target gene or a double strand of nucleic acid, wherein the sequence of the guide is at least 50%, 55%, 60%, 65%, 70% 80%, 85%, 90% or 95% complementarity, or may be a nucleotide sequence having complete complementarity.
  • the guiding sequence may be from 10 to 25 nucleotide sequences.
  • the guiding sequence may be from 10 to 25 nucleotides, from 15 to 25 nucleotides, or from 20 to 25 nucleotides.
  • the guide sequence may be from 10 to 15 nucleotides, from 15 to 20 nucleotides, or from 20 to 25 nucleotides.
  • the guiding domain may further comprise additional nucleotide sequences.
  • the additional nucleotide sequence may be for enhancing or reducing the function of the guide domain.
  • the additional nucleotide sequence may be for enhancing or reducing the function of the guide sequence.
  • the additional nucleotide sequence may be from 1 to 10 nucleotide sequences.
  • the additional nucleotide sequence may be from 2 to 10 nucleotides, from 4 to 10 nucleotides, from 6 to 10 nucleotides, or from 8 to 10 nucleotides.
  • the additional nucleotide sequence may be one to three nucleotide sequences, three to six nucleotide sequences, or seven to ten nucleotide sequences.
  • the additional nucleotide sequence comprises one nucleotide sequence, two nucleotide sequences, three nucleotide sequences, four nucleotide sequences, five nucleotide sequences, six nucleotide sequences, seven nucleotide sequences, eight nucleotide sequences, 9 nucleotide sequences or 10 nucleotide sequences.
  • the additional nucleotide sequence may be one nucleotide sequence G (guanine) or two nucleotide sequences GG.
  • the additional nucleotide sequence may be located at the 5 ' end of the guide sequence.
  • the additional nucleotide sequence may be located at the 3 ' end of the guide sequence.
  • a “first complementary domain” is a domain containing a complementary nucleotide sequence to a second complementary domain described below, and is complementary enough to form a double strand with a second complementary domain.
  • the first complementary domain may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% complementarity for the second complementary domain Or may be a nucleotide sequence having complete complementarity.
  • the first complementary domain may form a double strand through complementary binding with the second complementary domain.
  • the double strand formed may interact with some amino acids of the editor protein to form a guide nucleic acid-editor protein complex.
  • the first complementary domain may be from 5 to 35 nucleotide sequences.
  • the first complementary domain comprises a sequence of 5 to 35 nucleotides, 10 to 35 nucleotides, 15 to 35 nucleotides, 20 to 35 nucleotides, 25 to 35 nucleotides, or 30 to 35 nucleotides Lt; / RTI >
  • the first complementary domain comprises one to five nucleotide sequences, five to ten nucleotide sequences, 10 to 15 nucleotide sequences, 15 to 20 nucleotide sequences, 20 to 25 nucleotide sequences, 25 to 30 nucleotides, Lt; / RTI > nucleotide sequence or 30 to 35 nucleotide sequences.
  • a " connecting domain" is a nucleotide sequence that links two or more domains, and the connecting domain connects two or more identical or different domains.
  • a connection domain may have a covalent or non-covalent association with two or more domains, or two or more domains may be covalently or non-covalently linked.
  • the linking domain may be from 1 to 30 nucleotide sequences.
  • the linking domain comprises one to five nucleotide sequences, 5 to 10 nucleotide sequences, 10 to 15 nucleotide sequences, 15 to 20 nucleotide sequences, 20 to 25 nucleotide sequences, or 25 to 30 nucleotide sequences .
  • the linking domain comprises one to 30 nucleotide sequences, 5 to 30 nucleotide sequences, 10 to 30 nucleotide sequences, 15 to 30 nucleotide sequences, 20 to 30 nucleotide sequences, or 25 to 30 nucleotide sequences Lt; / RTI >
  • a “second complementary domain” is a domain containing a nucleotide sequence comprising a first complementary domain and a complementary nucleic acid sequence, and is complementary enough to form a double strand with the first complementary domain.
  • the second complementary domain may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% complementary to the first complementary domain Or may be a nucleotide sequence having complete complementarity.
  • the second complementary domain may form a double strand through complementary binding with the first complementary domain.
  • the double strand formed may interact with some amino acids of the editor protein to form a guide nucleic acid-editor protein complex.
  • the second complementary domain comprises a nucleotide sequence that is not complementary to the first complementary domain and the complementary nucleotide sequence and the first complementary domain, for example, a nucleotide sequence that does not form a double strand with the first complementary domain And the length of the nucleotide sequence may be longer than that of the first complementary domain.
  • the second complementary domain may be from 5 to 35 nucleotide sequences.
  • the second complementary domain comprises from 1 to 35 nucleotides, from 5 to 35 nucleotides, from 10 to 35 nucleotides, from 15 to 35 nucleotides, from 20 to 35 nucleotides, from 25 to 35 nucleotides, Sequence or a 30 to 35 nucleotide sequence.
  • the second complementary domain comprises one to five nucleotide sequences, 5 to 10 nucleotide sequences, 10 to 15 nucleotide sequences, 15 to 20 nucleotide sequences, 20 to 25 nucleotide sequences, 25 to 30 nucleotide sequences, Nucleotide sequence or 30 to 35 nucleotide sequences.
  • proximal domain is a nucleotide sequence located close to a second complementary domain.
  • Proximal domains may contain complementary nucleotide sequences within the proximal domain and may form double strands by complementary nucleotide sequences.
  • the proximal domain may be from 1 to 20 nucleotide sequences.
  • the proximal domain may be from 1 to 20 nucleotide sequences, from 5 to 20 nucleotide sequences, from 10 to 20 nucleotide sequences, or from 15 to 20 nucleotide sequences.
  • the proximal domain may be from 1 to 5 nucleotides, from 5 to 10 nucleotides, from 10 to 15 nucleotides, or from 15 to 20 nucleotides.
  • the " tail domain” is a nucleotide sequence located at one or more ends of the two ends of the guide nucleic acid.
  • the tail domain may comprise a complementary nucleotide sequence within the tail domain and may form a double strand by a complementary nucleotide sequence.
  • the tail domain may be from 1 to 50 nucleotide sequences.
  • the tail domain comprises 5 to 50 nucleotides, 10 to 50 nucleotides, 15 to 50 nucleotides, 20 to 50 nucleotides, 25 to 50 nucleotides, 30 to 50 nucleotides, 35 To 50 nucleotide sequences, from 40 to 50 nucleotide sequences, or from 45 to 50 nucleotide sequences.
  • the tail domain comprises one to five nucleotide sequences, five to ten nucleotide sequences, 10 to 15 nucleotide sequences, 15 to 20 nucleotide sequences, 20 to 25 nucleotide sequences, 25 to 30 nucleotide sequences, 30 to 35 nucleotide sequences, 35 to 40 nucleotide sequences, 40 to 45 nucleotide sequences, or 45 to 50 nucleotide sequences.
  • nucleic acid sequences including the above-mentioned domains i.e., the guide domain, the first complementary domain, the connecting domain, the second complementary domain, the proximal domain and the tail domain may optionally or additionally include a chemical modification have.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.
  • the guide nucleic acid comprises one or more domains.
  • the guide nucleic acid may comprise a guide domain.
  • the guide nucleic acid may comprise a first complementary domain.
  • the guide nucleic acid may comprise a linking domain.
  • the guide nucleic acid may comprise a second complementary domain.
  • the guide nucleic acid may comprise a proximal domain.
  • the guide nucleic acid may comprise a tail domain.
  • the number of the domains may be 1, 2, 3, 4, 5, 6, or more.
  • the guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more guide domains.
  • the guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more first complementary domains.
  • the guide nucleic acid may comprise one, two, three, four, five, six or more linking domains.
  • the guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more second complementary domains.
  • the guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more proximal domains.
  • the guide nucleic acid may comprise one, two, three, four, five, six or more tail domains.
  • the guide nucleic acid may include one domain in duplicate.
  • the guide nucleic acid may comprise multiple domains without overlapping or overlapping.
  • the guide nucleic acid may comprise the same kind of domain, wherein the same kind of domains may have the same nucleic acid sequence or may have different nucleic acid sequences.
  • the guide nucleic acid may comprise two kinds of domains, wherein the two other domains may have different nucleic acid sequences or may have the same nucleic acid sequence.
  • the guide nucleic acid may comprise three kinds of domains, wherein the three other domains may have different nucleic acid sequences or may have the same nucleic acid sequence.
  • the guide nucleic acid may comprise four kinds of domains, wherein the other four kinds of domains may have different nucleic acid sequences or may have the same nucleic acid sequences.
  • the guide nucleic acid may include five kinds of domains, wherein the other five kinds of domains may have different nucleic acid sequences or may have the same nucleic acid sequences.
  • the guide nucleic acid may comprise six kinds of domains, wherein the other six kinds of domains may have different nucleic acid sequences or may have the same nucleic acid sequence.
  • the guide nucleic acid may be selected from [guide domain] - [first complementary domain] - [link domain] - [second complementary domain] - [link domain] - [guide domain] - [first complementary domain] - [Linked domain] - [Second complementary domain], where the two guide domains may contain a guide sequence for different or identical targets, and the two first complementary domains Two second complementary domains may have the same nucleic acid sequence or have different nucleic acid sequences. Where the guiding domain comprises a guiding sequence for a different target, the guiding nucleic acid may specifically bind to two targets, wherein specific binding may occur simultaneously or sequentially.
  • the linkage domain can be cleaved by a specific enzyme, and in the presence of a specific enzyme, the guide nucleic acid can be divided into two parts or three parts.
  • the guide nucleic acid may be a gRNA.
  • gRNA &quot refers to a gRNA-CRISPR enzyme complex for the target gene or nucleic acid, i. E., An RNA capable of specifically targeting the CRISPR complex.
  • the gRNA means a target gene or a nucleic acid-specific RNA.
  • the gRNA can be coupled with a CRISPR enzyme to direct a CRISPR enzyme to a target gene or a nucleic acid.
  • the gRNA may comprise a plurality of domains. Each domain can interact within the strand or between strands of the three-dimensional behavior or the active form of the gRNA.
  • gRNA is a single stranded gRNA (single RNA molecule; single gRNA; sgRNA); Or a double gRNA (including more than one and typically two separate RNA molecules).
  • the single stranded gRNA comprises a guide domain in the 5 'to 3' direction, a domain comprising a guide sequence capable of binding complementary to a target gene or nucleic acid; A first complementary domain; Connection domain; A second complementary domain, a domain capable of forming a double-stranded nucleic acid with a first complementary domain since the first complementary domain has a sequence complementary to the first complementary domain sequence; Proximal domain; And optionally a tail domain.
  • the double gRNA comprises a guide domain in the 5 'to 3' direction, a domain comprising a guide sequence capable of a complementary binding to a target gene or nucleic acid, and a first complementary domain ≪ / RTI > And a second complementary domain; a domain capable of forming a double-stranded nucleic acid with a first complementary domain having a sequence complementary to the first complementary domain sequence; a proximal domain; And optionally a second strand comprising a tail domain.
  • first strand may be referred to as a crRNA and the second strand may be referred to as a tracrRNA.
  • the crRNA may comprise a guiding domain and a first complementary domain, wherein the tracrRNA may comprise a second complementary domain, a proximal domain and optionally a tail domain.
  • the single stranded gRNA comprises a guide domain in the 3 'to 5' direction, a domain comprising a guide sequence capable of binding complementary to the target gene or nucleic acid; A first complementary domain; And a second complementary domain, a domain complementary to the first complementary domain sequence, and a domain capable of forming a double-stranded nucleic acid with the first complementary domain.
  • the first complementary domain may have homology with the first complementary domain derived from nature, or may be derived from a first complementary domain derived from nature.
  • the first complementary domain may have a nucleotide sequence different from that of the first complementary domain depending on the species present in nature, and may be derived from a first complementary domain comprising the species present in nature, or And may have some or complete homology with the first complementary domain comprising the species present in nature.
  • the first complementary domain Streptococcus blood yoge Ness (Streptococcus pyogenes), Campylobacter Jeju Needle (Campylobacter jejuni), Streptococcus Thermo filler's (Streptococcus thermophiles), Staphylococcus aureus (Staphylococcus at least 50%, or complete homology with the first complementary domain or the derived first complementary domain of aureus or Neisseria meningitides .
  • the first complementary domain when the first complementary domain is a first complementary domain of Streptococcus fyogenes or a first complementary domain derived from Streptococcus fyogenses, the first complementary domain is 5'-GUUUUAGAGCUA-3 Or may be a nucleotide sequence having at least 50% or more homology with 5'-GUUUUAGAGCUA-3 '.
  • the first complementary domain may further include (X) n , i.e., 5'-GUUUUAGAGCUA (X) n -3 '.
  • X may be selected from the group consisting of nucleotides A, T, U, and G, wherein n is the number of nucleotide sequences and may be an integer of 5 to 15.
  • (X) n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • the first complementary domain when the first complementary domain is a first complementary domain of Campylobacter jejuni or a first complementary domain of Campylobacter herbaceae, the first complementary domain is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU -3 'or 5'-GUUUUAGUCCCUU-3', or may be a nucleotide sequence having at least 50% or more homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3 'or 5'-GUUUUAGUCCCUU-3'.
  • the first complementary domain may further include (X) n , that is, 5'-GUUUUAGUCUCUUUUUAAAUUUCUU (X) n -3 'or 5'-GUUUUAGUCCCUU (X) n -3'.
  • X may be selected from the group consisting of nucleotides A, T, U, and G, wherein n is the number of nucleotide sequences and may be an integer of 5 to 15.
  • (X) n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • the first complementary domain is selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus , , Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae , Lachnospiraceae bacterium (ND2006), Pseudomonas spp. ), Porphyromonas crevioricanis , Prevotella disiens , Moraxella bovoculi (237), Smiihella sp.
  • the first complementary domain when the first complementary domain is a first complementary domain of P. bacterium or a first complementary domain of P. bacterium, the first complementary domain is 5'-UUUGUAGAU-3 ' Or may be a nucleotide sequence having at least 50% or more homology with 5'-UUUGUAGAU-3 '.
  • the first complementary domain comprises (X) n in addition, that is, 5 - may '(X) n UUUGUAGAU-3 '.
  • X may be selected from the group consisting of nucleotides A, T, U, and G, wherein n is the number of nucleotide sequences and may be an integer of 1 to 5.
  • (X) n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • the connecting domain may be a nucleotide sequence which serves to connect the first complementary domain and the second complementary domain.
  • the connecting domain may have a covalent bond or a non-covalent bond with the first complementary domain and the second complementary domain, respectively.
  • connection domain may connect the first complementary domain and the second complementary domain in a covalent or non-covalent manner.
  • the linking domain is suitable for use in single-stranded gRNA molecules, and can be covalently or non-covalently linked to the first and second strands of the double gRNA, or covalently or non-covalently linked the first and second strands Can be used to generate single stranded gRNA.
  • the linking domain can be used for covalent or noncovalent binding with the crRNA and tracrRNA of the double gRNA, or covalently or noncovalently linking the crRNA and the tracrRNA to produce single stranded gRNA.
  • the second complementary domain may have a homology with the second complementary domain derived from nature, or may be derived from a second complementary domain derived from nature.
  • the second complementary domain may have a difference in the nucleotide sequence of the second complementary domain depending on the species present in nature, and may be derived from a second complementary domain comprising the species present in nature, or May have some or complete homology with a second complementary domain comprising the species present in nature.
  • the second complementary domain Streptococcus blood yoge Ness (Streptococcus pyogenes), Campylobacter Jeju Needle (Campylobacter jejuni), Streptococcus Thermo filler's (Streptococcus thermophiles), Staphylococcus aureus (Staphylococcus at least 50%, or complete homology with the second complementary domain or the derived second complementary domain of aureus or Neisseria meningitides .
  • the second complementary domain is a second complementary domain of Streptococcus fyogenes or a second complementary domain of Streptococcus fyogenses
  • the second complementary domain is 5'- UAGC AAGU UAAAA U-3 ', or may be a nucleotide sequence having at least 50% or more homology with the 5'- UAGC AAGU UAAAA U-3' (underlined indicates that the first complementary domain forms a double strand Nucleotide sequence).
  • the second complementary domain may further include (X) n or / and (X) m , that is, 5 '- (X) n UAGC AAGU UAAAA U (X) m -3'.
  • X may be selected from the group consisting of nucleotides A, T, U and G, wherein n and m are the number of nucleotide sequences, n may be an integer of 1 to 15, and m is an integer of 1 to 6 have.
  • n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • (X) m can be an integer number m of repetitions of the same nucleotide sequence or an integer number m of nucleotide sequences in which the nucleotides A, T, U and G are mixed.
  • the second complementary domain when the second complementary domain is a second complementary domain of Campylobacter jejuni or a second complementary domain of Campylobacter zeaxin , the second complementary domain is 5'- AAGAAAUUUAAAAAAGGGACUAAAA U-3 'or 5'- AAGGGACUAAAA U-3', or may be a nucleotide sequence having at least 50% or more homology with some of 5'- AAGAAAUUUAAAAAGGGACUAAAA U-3 'or 5'- AAGGGACUAAAA U-3' (Underlined is a nucleotide sequence forming a first complementary domain and a double strand).
  • the second complementary domain comprises adding to (X) n and / or (X) m, i.e., 5 '- (X) n AAGAAAUUUAAAAAGGGACUAAAA U (X) m -3' or 5 '- (X) n AAGGGACUAAAA U (X) m -3 '.
  • X may be selected from the group consisting of nucleotides A, T, U and G
  • n may be an integer of 1 to 15, and m may be 1 to 6.
  • (X) n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • (X) m can be an integer number m of repetitions of the same nucleotide sequence or an integer number m of nucleotide sequences in which the nucleotides A, T, U and G are mixed.
  • the second complementary domain is selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus , , Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae , Lachnospiraceae bacterium (ND2006), Pseudomonas spp. ), Porphyromonas crevioricanis , Prevotella disiens , Moraxella bovoculi (237), Smiihella sp.
  • the second complementary domain is a second complementary domain of P. bacterium or a second complementary domain of P. bacterium
  • the second complementary domain is 5'-AAAUU UCUAC U-3
  • the second complementary domain may be a nucleotide sequence having at least 50% or more homology with 5'-AAAUU UCUAC U-3 '(underlined is the nucleotide sequence forming a double strand with the first complementary domain).
  • the second complementary domain may further include (X) n or / and (X) m , that is, 5 '- (X) n AAAUU UCUAC U (X) m -3'.
  • X may be selected from the group consisting of nucleotides A, T, U and G, wherein n and m are the number of nucleotide sequences, n may be an integer of 1 to 10, and m is an integer of 1 to 6 have.
  • n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • (X) m can be an integer number m of repetitions of the same nucleotide sequence or an integer number m of nucleotide sequences in which the nucleotides A, T, U and G are mixed.
  • the first complementary domain and the second complementary domain may have a complementary combination.
  • the first complementary domain and the second complementary domain may form double strands through the complementary binding.
  • the double strand thus formed can interact with the CRISPR enzyme.
  • the first complementary domain may comprise an additional nucleotide sequence that does not undergo a complementary binding with the second complementary domain of the second strand.
  • the additional nucleotide sequence may be from 1 to 15 nucleotide sequences.
  • the additional nucleotide sequence may be from 1 to 5 nucleotide sequences, from 5 to 10 nucleotide sequences, or from 10 to 15 nucleotide sequences.
  • the proximal domain may be a domain located in the 3 'direction of the second complementary domain.
  • the proximal domain may have homology with a naturally occurring proximal domain or may be derived from a naturally occurring proximal domain.
  • the proximal domain is selected from the group consisting of Streptococcus pyogenes , Campylobacter jejuni , Streptococcus thermophiles , Staphylococcus aureus , or Staphylococcus aureus . At least 50%, or complete homology with the proximal or derived proximal domain of Neisseria meningitides .
  • the proximal domain when the proximal domain is a proximal domain of Streptococcus fjigogenes or a proximal domain derived from Streptococcus fjigenses, the proximal domain may be 5'-AAGGCUAGUCCG-3 ', or 5'-AAGGCUAGUCCG-3' And a nucleotide sequence having at least 50% homology with the nucleotide sequence of SEQ ID NO. At this time, the proximal domain may further include (X) n , that is, 5'-AAGGCUAGUCCGG (X) n -3 '.
  • X may be selected from the group consisting of nucleotides A, T, U, and G, wherein n is the number of nucleotide sequences and may be an integer of 1 to 15. (X) n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • the proximal domain may be 5'-AAAGAGUUUGC-3 ', or 5'-AAAGAGUUUGC -3 'and at least 50% homology with the nucleotide sequence of SEQ ID NO: 2.
  • the proximal domain may further include (X) n , i.e., 5'-AAAGAGUUUGC (X) n -3 '.
  • X may be selected from the group consisting of nucleotides A, T, U, and G, wherein n is the number of nucleotide sequences and may be an integer of 1 to 40.
  • n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • the tail domain can be selectively added to the 3 ' end of the single strand gRNA or the first strand or the second strand of the double gRNA.
  • the tail domain may have a homology with a naturally occurring tail domain, or may be derived from a naturally occurring tail domain.
  • the tail domain is selected from the group consisting of Streptococcus pyogenes , Campylobacter jejuni , Streptococcus thermophiles , Staphylococcus aureus or At least 50%, or complete homology with the tail domain or the derived tail domain of Neisseria meningitides .
  • the tail domain may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 ', or 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 And a nucleotide sequence having at least 50% homology with the nucleotide sequence of SEQ ID NO.
  • the tail domain may further include (X) n , i.e., 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (X) n -3 '.
  • X may be selected from the group consisting of nucleotides A, T, U, and G, wherein n is the number of nucleotide sequences and may be an integer of 1 to 15. (X) n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • the tail domain may be 5'-GGGACUCUGCGGGUUACAAUCCCCUAAAACCGCUUU-3 ', or 5'-GGGACUCUGCGGGUUACAAUCCCCUAAAACCGCUUUU -3 'and at least 50% homology with the nucleotide sequence of SEQ ID NO: 2.
  • the tail domain may further include (X) n , that is, 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU (X) n -3 '.
  • X may be selected from the group consisting of nucleotides A, T, U, and G, wherein n is the number of nucleotide sequences and may be an integer of 1 to 15. (X) n may be an integer number n of repeats of the same nucleotide sequence or an integer number n of nucleotide sequences in which the nucleotides A, T, U, and G are mixed.
  • the tail domain may comprise from 1 to 10 nucleotide sequences at the 3 ' end associated with in vitro or in vivo transcription methods.
  • the tail domain when a T7 promoter is used for in vitro transcription of a gRNA, the tail domain may be any nucleotide sequence present at the 3 ' end of the DNA template.
  • the tail domain when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU, and when the H1 promoter is used for transcription, the tail domain may be UUUU, and when the pol-III promoter is used ,
  • the tail domain may comprise several uracil nucleotides or alternatively nucleotides.
  • the gRNA may include a plurality of domains as described above, and the length of the nucleic acid sequence can be controlled according to the type and number of the domains contained in the gRNA, and the three-dimensional form or the active form of the gRNA Interactions within the strands or between the strands.
  • gRNA is a single stranded gRNA (single RNA molecule); Or a double gRNA (including more than one and typically two separate RNA molecules).
  • the double gRNA consists of a first strand and a second strand.
  • first strand may be referred to as a crRNA and the second strand may be referred to as a tracrRNA.
  • first strand and the second strand may optionally comprise additional nucleotide sequences.
  • the first strand comprises
  • the N target is a nucleotide sequence complementary to a partial sequence of a target gene or a double strand of nucleic acid
  • the N target is a nucleotide sequence region which can be changed according to a target sequence on a target gene or a nucleic acid.
  • (Q) m comprises a nucleotide sequence comprising a first complementary domain and a nucleotide sequence capable of complementary binding to a second complementary domain of a second strand.
  • the (Q) m may be a sequence having partial or complete homology with the first complementary domain of the species present in nature, and the nucleotide sequence of the first complementary domain may be changed depending on the species derived.
  • the Q may be independently selected from the group consisting of A, U, C and G, and m is the number of the nucleotide sequence, and may be an integer of 5 to 35.
  • the (Q) m May be 5'-GUUUUAGAGCUA-3 ', or may be a nucleotide sequence having at least 50% homology with 5'-GUUUUAGAGCUA-3'.
  • the (Q) m is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3 'or 5'-GUUUUAGUCCCUU-3', or may be a nucleotide sequence having at least 50% homology with 5'-GUUUUAGUCUCUUUUUAAAUUUCUU-3 'or 5'-GUUUUAGUCCCUU-3'.
  • the (Q) m May be 5'-GUUUUAGAGCUGUGUUGUUUCG-3 ', or may be a nucleotide sequence having at least 50% or more homology with 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.
  • (X) a , (X) b and (X) c are nucleotide sequences that can be optionally added, wherein X may be independently selected from the group consisting of A, U, C and G, A, b, and c are the number of nucleotide sequences and may be 0 or an integer of 1 to 20.
  • the second strand comprises
  • the second strand comprises
  • (Z) h is a nucleotide sequence including a second complementary domain, and includes a nucleotide sequence capable of complementary binding with a first complementary domain of the first strand.
  • the (Z) h may be a sequence having partial or complete homology with the second complementary domain of the species present in nature, and the nucleotide sequence of the second complementary domain may be altered depending on the derived species.
  • Z may be independently selected from the group consisting of A, U, C and G, and h is the number of nucleotide sequences, and may be an integer of 5 to 50.
  • the (Z) h May be 5'-UAGCAAGUUAAAAU-3 ', or may be a nucleotide sequence having at least 50% or more homology with 5'-UAGCAAGUUAAAAU-3'.
  • the (Z) h is 5'-AAGAAAUUUAAAAAGGGACUAAAA-3 'or 5'-AAGGGACUAAAAU-3', or may be a nucleotide sequence having at least 50% or more homology with 5'-AAGAAAUUUAAAAAGGGACUAAAA-3 'or 5'-AAGGGACUAAAAU-3'.
  • the (Z) h May be 5'-CGAAACAACACAGCGAGUUAAAAU-3 ', or may be a nucleotide sequence having at least 50% or more homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3'.
  • (P) k is a nucleotide sequence comprising a proximal domain, and may be a sequence having partial or complete homology with a proximal domain of a species present in nature, and the nucleotide sequence of the proximal domain may be altered .
  • P may be independently selected from the group consisting of A, U, C and G, and k is the number of nucleotide sequences and may be an integer of 1 to 20.
  • (P) k is 5'-AAGGCUAGUCCG-3 '
  • (P) k may be a nucleotide sequence having at least 50% or more homology with 5'-AAGGCUAGUCCG-3 '.
  • (P) k may be 5'-AAAGAGUUUGC-3 'when the proximal domain has some or complete homology with the proximal domain of Campylobacter jejuni or Campylobacter sp. , Or a nucleotide sequence having at least 50% or more homology with 5'-AAAGAGUUUGC-3 '.
  • (P) k is 5'-AAGGCUUAGUCCG-3 '
  • (P) k may be a nucleotide sequence having at least 50% or more homology with 5'-AAGGCUUAGUCCG-3 '.
  • (F) i is a nucleotide sequence comprising a tail domain, and may be a sequence having partial or complete homology with the tail domain of a species present in nature, and the nucleotide sequence of the tail domain may be altered depending on the species derived .
  • the F may be independently selected from the group consisting of A, U, C and G, wherein i is the number of the nucleotide sequence and may be an integer of 1 to 50.
  • (F) i is 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 '
  • (F) i is 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 '
  • the (F) i may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 '
  • the (F) i may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 '
  • tail domain has partial or complete homology with the tail domain of Streptococcus thermophilus or the tail domain from Streptococcus thermophilus
  • (F) i is 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUU-3 '
  • (F) i may comprise 1 to 10 nucleotide sequences at the 3 'end associated with in vitro or in vivo transcription methods.
  • the tail domain when a T7 promoter is used for in vitro transcription of a gRNA, the tail domain may be any nucleotide sequence present at the 3 ' end of the DNA template.
  • the tail domain when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU, and when the H1 promoter is used for transcription, the tail domain may be UUUU, and when the pol-III promoter is used ,
  • the tail domain may comprise several uracil nucleotides or alternatively nucleotides.
  • (X) d , (X) e and (X) f are nucleotide sequences that can be selectively added, and X may be independently selected from the group consisting of A, U, C and G, D, e, and f are the number of nucleotide sequences and may be 0 or an integer of 1 to 20.
  • Single stranded gRNA can be divided into a first single stranded gRNA and a second single stranded gRNA.
  • the first single-stranded gRNA is a single-stranded gRNA that connects the first and second strands of the double gRNA to the connecting domain.
  • the single stranded gRNA comprises
  • the first single-stranded gRNA may optionally comprise additional nucleotide sequences.
  • the first single stranded gRNA comprises
  • the single stranded gRNA comprises
  • the N target is a nucleotide sequence complementary to a partial sequence of a target gene or a double strand of nucleic acid
  • the N target is a nucleotide sequence region which can be changed according to a target sequence on a target gene or a nucleic acid.
  • (Q) m comprises a nucleotide sequence comprising a first complementary domain, and a nucleotide sequence capable of complementary binding to a second complementary domain.
  • the (Q) m may be a sequence having partial or complete homology with the first complementary domain of the species present in nature, and the nucleotide sequence of the first complementary domain may be changed depending on the species derived.
  • the Q may be independently selected from the group consisting of A, U, C and G, and m is the number of the nucleotide sequence, and may be an integer of 5 to 35.
  • the (Q) m May be 5'-GUUUUAGAGCUA-3 ', or may be a nucleotide sequence having at least 50% homology with 5'-GUUUUAGAGCUA-3'.
  • the (Q) m is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3 'or 5'-GUUUUAGUCCCUU-3', or may be a nucleotide sequence having at least 50% homology with 5'-GUUUUAGUCUCUUUUUAAAUUUCUU-3 'or 5'-GUUUUAGUCCCUU-3'.
  • the (Q) m May be 5'-GUUUUAGAGCUGUGUUGUUUCG-3 ', or may be a nucleotide sequence having at least 50% or more homology with 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.
  • (L) j is a nucleotide sequence including a linking domain, and is a nucleotide sequence that allows a first complementary domain and a second complementary domain to be joined to produce single stranded gRNA.
  • the L may be independently selected from the group consisting of A, U, C, and G, and j is the number of nucleotide sequences and may be an integer of 1 to 30.
  • (Z) h is a nucleotide sequence comprising a second complementary domain, and comprises a nucleotide sequence capable of complementary binding with a first complementary domain.
  • the (Z) h may be a sequence having partial or complete homology with the second complementary domain of the species present in nature, and the nucleotide sequence of the second complementary domain may be altered depending on the derived species.
  • Z may be independently selected from the group consisting of A, U, C and G, and h is the number of nucleotide sequences, and may be an integer of 5 to 50.
  • the (Z) h May be 5'-UAGCAAGUUAAAAU-3 ', or may be a nucleotide sequence having at least 50% or more homology with 5'-UAGCAAGUUAAAAU-3'.
  • the (Z) h is 5'-AAGAAAUUUAAAAAGGGACUAAAA-3 'or 5'-AAGGGACUAAAAU-3', or may be a nucleotide sequence having at least 50% or more homology with 5'-AAGAAAUUUAAAAAGGGACUAAAA-3 'or 5'-AAGGGACUAAAAU-3'.
  • the (Z) h May be 5'-CGAAACAACACAGCGAGUUAAAAU-3 ', or may be a nucleotide sequence having at least 50% or more homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3'.
  • (P) k is a nucleotide sequence comprising a proximal domain, and may be a sequence having partial or complete homology with a proximal domain of a species present in nature, and the nucleotide sequence of the proximal domain may be altered .
  • P may be independently selected from the group consisting of A, U, C and G, and k is the number of nucleotide sequences and may be an integer of 1 to 20.
  • (P) k is 5'-AAGGCUAGUCCG-3 '
  • (P) k may be a nucleotide sequence having at least 50% or more homology with 5'-AAGGCUAGUCCG-3 '.
  • (P) k may be 5'-AAAGAGUUUGC-3 'when the proximal domain has some or complete homology with the proximal domain of Campylobacter jejuni or Campylobacter sp. , Or a nucleotide sequence having at least 50% or more homology with 5'-AAAGAGUUUGC-3 '.
  • (P) k is 5'-AAGGCUUAGUCCG-3 '
  • (P) k may be a nucleotide sequence having at least 50% or more homology with 5'-AAGGCUUAGUCCG-3 '.
  • (F) i is a nucleotide sequence comprising a tail domain, and may be a sequence having partial or complete homology with the tail domain of a species present in nature, and the nucleotide sequence of the tail domain may be altered depending on the species derived .
  • the F may be independently selected from the group consisting of A, U, C and G, wherein i is the number of the nucleotide sequence and may be an integer of 1 to 50.
  • (F) i is 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 '
  • (F) i is 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 '
  • the (F) i may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 '
  • the (F) i may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 '
  • tail domain has partial or complete homology with the tail domain of Streptococcus thermophilus or the tail domain from Streptococcus thermophilus
  • (F) i is 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUU-3 '
  • (F) i may comprise 1 to 10 nucleotide sequences at the 3 'end associated with in vitro or in vivo transcription methods.
  • the tail domain when a T7 promoter is used for in vitro transcription of a gRNA, the tail domain may be any nucleotide sequence present at the 3 ' end of the DNA template.
  • the tail domain when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU, and when the H1 promoter is used for transcription, the tail domain may be UUUU, and when the pol-III promoter is used ,
  • the tail domain may comprise several uracil nucleotides or alternatively nucleotides.
  • the (X) a, (X) b, (X) c, (X) d, (X) e and (X) f is a nucleotide sequence that can be optionally added, wherein X is A, U, C, and G, wherein a, b, c, d, e, and f are the number of nucleotide sequences and may be 0 or an integer of 1 to 20.
  • the second single-stranded gRNA may be a single-stranded gRNA consisting of a guiding domain, a first complementary domain and a second complementary domain.
  • the second single-stranded gRNA may optionally comprise additional nucleotide sequences.
  • the second single-stranded gRNA comprises
  • the single stranded gRNA comprises
  • the N target is a nucleotide sequence complementary to a partial sequence of a target gene or a double strand of nucleic acid
  • the N target is a nucleotide sequence region which can be changed according to a target sequence on a target gene or a nucleic acid.
  • (Q) m comprises a nucleotide sequence comprising a first complementary domain, and a nucleotide sequence capable of complementary binding to a second complementary domain.
  • the (Q) m may be a sequence having partial or complete homology with the first complementary domain of the species present in nature, and the nucleotide sequence of the first complementary domain may be changed depending on the species derived.
  • the Q may be independently selected from the group consisting of A, U, C and G, and m is the number of the nucleotide sequence, and may be an integer of 5 to 35.
  • (Q) m is 5 '-UUUGUAGAU-3', or may be a nucleotide sequence having at least 50% homology with 5'-UUUGUAGAU-3 '.
  • (Z) h is a nucleotide sequence comprising a second complementary domain, and comprises a nucleotide sequence capable of complementary binding with a first complementary domain.
  • the (Z) h may be a sequence having partial or complete homology with the second complementary domain of the species present in nature, and the nucleotide sequence of the second complementary domain may be altered depending on the derived species.
  • Z may be independently selected from the group consisting of A, U, C and G, and h is the number of nucleotide sequences, and may be an integer of 5 to 50.
  • (Z) h is 5 '-AAAUUUCUACU-3', or may be a nucleotide sequence having at least 50% or more homology with 5'-AAAUUUCUACU-3 '.
  • (L) j is a nucleotide sequence including a linking domain, and is a nucleotide sequence linking a first complementary domain and a second complementary domain.
  • the L may be independently selected from the group consisting of A, U, C, and G, and j is the number of nucleotide sequences and may be an integer of 1 to 30.
  • (X) a , (X) b and (X) c are nucleotide sequences that can be optionally added, wherein X may be independently selected from the group consisting of A, U, C and G, A, b, and c are the number of nucleotide sequences and may be 0 or an integer of 1 to 20.
  • the guide nucleic acid may be a gRNA that is capable of complementarily binding to a target sequence of a retinal function-forming gene.
  • Retinal function-forming &quot refers to the function of the retina, for example, light entering the eye through the inner layer of the retina is sensed by the retina's photoreceptor, and the photoreceptor senses the light energy or signal by electrical energy or signal And all the functions necessary to transfer this energy or signal to the optic nerve through the cells of the retinal inner layer to function normally.
  • retinal function formation may be caused by various layers or membranes forming retinal tissue such as retinal pigment epithelium layer, photoreceptor cell layer, outer boundary layer, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, , The inner boundary layer, and the like, so that each layer or films can function normally.
  • retinal tissue such as retinal pigment epithelium layer, photoreceptor cell layer, outer boundary layer, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, , The inner boundary layer, and the like, so that each layer or films can function normally.
  • Retinal function formation also includes all processes that allow the functions of the various tissues and / or cells involved in retinal tissue to function normally to function normally.
  • the various tissues and / or cells involved in the retinal tissue to perform a normal function may be a tissue close to the retinal tissue, such as a choroid, a cornea, a sclera, an iris, a stroma, a ciliary body, etc. and / Cells.
  • retinal function formation also includes overall development and growth processes such as retinal cell survival, proliferation, persistence, and mission.
  • the retinal cells include neurons, glial cells, cone cells, glandular cells, retinal pigment epithelial cells, and the like.
  • Retinal function formation may be a function that senses the light of cone cells and / or stomach cells.
  • Retinal function formation may be a visual cycle related function.
  • the visual circuit is a circulatory process of photochemical change and regeneration of rhodopsin, a visual receptor, and involves the process of converting 11-cis-retinol into all-trans by photosynthesis in rhodopsin.
  • Retinal function formation may be modulation of the expression of visual cycle related genes and / or proteins.
  • Retinal function formation may be a function related to visual pigment regeneration.
  • &quot retinal function-forming gene " refers to any gene that directly participates in or indirectly influences the function of the retina, wherein the gene is directly involved in the functional formation of the retina, Encryption of all peptides, polypeptides or proteins that have an indirect effect includes both gene or nucleic acid sequences.
  • the retinal function-forming gene may be a wildtype retinal function-forming gene.
  • the retinal function-forming gene may be a retinal function-forming gene comprising one or more mutations.
  • &quot refers to a change in the nucleotide sequence of a DNA contained in a gene, and deletion, insertion or substitution of one or more nucleotides in the nucleotide sequence of the DNA contained in the gene, .
  • sequence in which a mutation occurs in a gene is referred to as a " mutation sequence ".
  • the mutation includes all of the amino acids of the protein encoded by the gene due to mutations in the gene, which have been modified by deletion, insertion or substitution.
  • the retinal function-forming gene is the RPE65 gene
  • the RPE65 gene includes the wildtype RPE65 gene and all naturally occurring mutant forms of the RPE65 gene.
  • the naturally occurring mutations of the RPE65 gene found to date are known to be 86, and the RPE65 gene disclosed herein includes all the above mutations.
  • the RPE65 gene is a naturally occurring mutant of the RPE65 gene, IVS-2A> C, IVS-2A> T, IVS2 + 1G> T, c.57_84delGG, c90_91insT, c.137delG, c106_114del9bp, c.292_311del20bp, c IVS8 + 1G> A, IVS8 + 4G> T, IVS6-1G> T, IVS6 + 1G> C, c.615_616delCA, IVS7 + 4A> G, IVS8 + , c.894delG, c.1064delA, c.1120delA, c.961_962insA, IVS11 + 2T> A, c.1056G> A, c.1059_1060insG, c.1069_1070insT, c.1243 + 2T> A, c.
  • the RPE65 gene is a naturally occurring M1T, E6 deletion, G32V, R44Q, L60P, Q64 deletion, F70V, R91Q, R91P, T101I, G104D, R118S, Y144D, E148D, T162P, H182R, H182N, N205S, D215G , R234 deletion, Y239D, Y249C, V287F, K303 deletion, H313R, Y318N, N321K, C330Y, L343 deletion, A360P, Y368C, A393G, W402 deletion, L403P, V407A, L408P, E417Q, Y431C, A434V, Y435C, G436V, , W458 deletion, W460 deletion, E462 deletion, P470L, G528V, F530L or S533T mutant RPE65 protein.
  • the one or more mutations may increase or decrease the expression of the protein encoded by the retinal function-forming gene.
  • the at least one mutation may be a synonymous mutation that does not affect the expression of the protein encoded by the retinal function-forming gene.
  • the at least one mutation may be a nonsense mutation that prevents the expression of a protein encoded by a retinal function-forming gene.
  • the retinal function-forming gene may promote or increase retinal function formation.
  • the retinal function-forming gene may inhibit or inhibit retinal function formation.
  • the retinal function-forming gene may induce or activate the retinal function-forming environment.
  • the retinal function-forming gene may induce an inhibitory environment of retinal function formation or deactivate the neovascularization environment.
  • the retinal function-forming gene may modulate (promote, increase, inhibit and / or inhibit) retinal function formation.
  • the retinal function-forming gene may express a protein that functions to sense light of cone cells and / or luminal cells.
  • the retinal function-forming gene may promote or increase the function of sensing the light of the cone and / or the liver cell.
  • the retinal function-forming gene may inhibit or inhibit the light-sensing function of cone cells and / or ganglion cells.
  • the retinal function-forming gene may promote or increase visual cycle-related functions.
  • the retinal function-forming gene may inhibit or inhibit visual cycle-related functions.
  • the retinal function-forming gene may express a protein that performs a visual cycle-related function.
  • the retinal function-forming gene can regulate the expression of a gene and / or protein that performs a visual cycle-related function.
  • the retinal function-forming gene may express a protein that performs a visual pigment regeneration-related function.
  • the retinal function-forming gene may promote or increase visual pigment regeneration-related functions.
  • the retinal function-forming gene may inhibit or inhibit visual pigment regeneration-related functions.
  • the retinal function-forming gene can be used for the improvement and treatment of retinal dysfunction.
  • the retinal function-forming gene disclosed in the present specification is a group consisting of RPE65 gene, GUCY2D gene, SPATA7 gene, AIPL1 gene, LCA5 gene, RPGRIP1 gene, CRB1 gene, CEP290 gene, IMPDH1 gene, RD3 gene, RDH12 gene and CRX gene It may be more than one selected.
  • the retinal function forming gene may be the RPE65 gene.
  • the RPE65 (retinal pigment epithelium-specific 65 kDa protein) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein RPE65, also referred to as BCO3, LCA2, RP20 or mrd12.
  • the RPE65 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human RPE65 (e.g., NCBI Accession No. NP_000320, etc.) RPE65 gene represented by NM_000329 and the like.
  • the RPE65 gene can be selected from the group consisting of IVS-2A> C, IVS-2A> T, IVS2 + 1G> T, c.57_84delGG, c90_91insT, c.137delG, c106_114del9bp, c.292_311del20bp, c.440_441delCA, c.495_496insG, IVS6-2A> T IVS8 + 1G> A, IVS8 + 4A> G, c.891delA, c.894delG, c.1064delA, c.1120delA, IVS6-1G> T, IVS6 + 1G> C, c.615_616delCA, IVS7 + , c.961_962insA, IVS11 + 2T> A, c.1056G> A, c.1059_1060insG, c.1069
  • the RPE65 gene has a deletion function of M1T, E6 deletion, G32V, R44Q, L60P, Q64 deletion, F70V, R91Q, R91P, T101I, G104D, R118S, Y144D, E148D, T162P, H182R, H182N, N205S, D215G, R234 deletion, Y239D, Y249C, Deletion, W460 deletion, W402 deletion, L403P, V407A, L408P, E417Q, Y431C, A434V, Y435C, G436V, V443A, W458 deletion, W460 deletion, E462 deletion, A360P, Y318Y, L343 deletion, A360P, Y368C, deletion, a mutant RPE65 gene encoding an RPE65 protein mutation such as P470L, G528V, F530L or S533T.
  • the retinal function forming gene may be the GUCY2D gene.
  • GUCY2D (guanylate cyclase 2D) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein GUCY2D, also referred to as CORD5, CORD6, CYGD, GUC1A4, GUC2D, LCA1, RCD2, RETGC-1, ROS-GC1 or ROSGC do.
  • the GUCY2D gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human GUCY2D (e.g., NCBI Accession No. NP - 000171, etc. GUCY2D gene expressed as NM_000180.
  • the retinal function-forming gene may be the SPATA7 gene.
  • the SPATA7 (Spermatogenesis-associated protein 7) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein SPATA7, also referred to as HSD3 or LCA3.
  • the SPATA7 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human SPATA7 (e.g., NCBI Accession No. NP_001035518, NP_060888, etc.) SPATA7 gene represented by NM_001040428, NM_018418, and the like.
  • the retinal function-forming gene may be the AIPL1 gene.
  • the AIPL1 (Aryl-hydrocarbon-interacting protein-like 1) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a protein AIPL1 also called LCA4.
  • the AIPL1 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human AIPL1 (e.g., NCBI Accession No. NP_001028226, NP_001028227, etc.) NM_014336, NM_001033054, and the like.
  • the retinal function-forming gene may be the LCA5 gene.
  • the LCA5 (leber congenital amaurosis 5) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein LCA5, also called lebercilin or C6orf152.
  • the LCA5 gene can be, but is not limited to, one or more selected from the group consisting of: a gene encoding human LCA5 (e.g., NCBI Accession No. NP_001116241, NP_859065, etc.)
  • the LCA5 gene represented by NM_001122769, NM_181714, and the like.
  • the retinal function forming gene may be the RPGRIP1 gene.
  • the gene for RPGRIP1 (X-linked retinitis pigmentosa GTPase regulator-interacting protein 1) refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein RPGRIP1 also called CORD13, LCA6, RGI1, RGRIP, RPGRIP or RPGRIP1d.
  • the RPGRIP1 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human RPGRIP1 (e.g., NCBI Accession No. NP_065099, etc.) RPGRIP1 gene represented by NM_020366.
  • the retinal function-forming gene may be the CRB1 gene.
  • the CRB1 (Crumbs homolog 1) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein CRB1, which is also referred to as LCA8 or RP12.
  • the CRB1 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human CRB1 (e.g., NCBI Accession No. NP_001180569, NP_001244894, etc.) CRB1 gene represented by NM_001193640, NM_001257965, and the like.
  • the retinal function forming gene may be the CEP290 gene.
  • the CEP290 (Centrosomal protein of 290 kDa) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein CEP290, also referred to as BBS14, CT87, JBTS5 or LCA10.
  • the CEP290 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human CEP290 (e.g., NCBI Accession No. NP - 079390); CEP290 gene represented by NM_025114.
  • the retinal function forming gene may be the IMPDH1 gene.
  • IMPDH1 (Inosine-5'-monophosphate dehydrogenase 1) gene refers to a gene (full-length DNA, cDNA or mRNA) which encodes a protein IMPDH1 also called IMPD1, IMPDH-I, LCA11 or RP10.
  • the IMPDH1 gene can be, but is not limited to, one or more selected from the group consisting of: a gene encoding human IMPDH1 (e.g., NCBI Accession No. NP - 000874, NP - 001096075, etc.
  • the IMPDH1 gene represented by NM_000883, NM_001102605, and the like.
  • the retinal function forming gene may be the RD3 gene.
  • the RD3 (Retinal Degeneration 3) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a protein RD3, also referred to as LCA12 or C1orf36.
  • the RD3 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human RD3 (eg, NCBI Accession No. NP_001158160.1, NP_898882.1, etc.) Accession No. RD3 gene represented by NM_001164688.1, NM_183059.2, and the like.
  • the retinal function forming gene may be the RDH12 gene.
  • the RDH12 (Retinol dehydrogenase 12) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein RDH12, also referred to as LCA13, RP53 or SDR7C2.
  • the RDH12 gene may be, but is not limited to, one or more selected from the group consisting of: a gene encoding human RDH12 (e.g., NCBI Accession No. NP - 689656) RDH12 gene represented by NM_152443.
  • the retinal function forming gene may be a CRX gene.
  • the CRX (Cone-rod homeobox protein) gene refers to a gene (full-length DNA, cDNA or mRNA) that encodes a protein CRX, also referred to as CORD2, CRD, LCA7 or OTX3.
  • the CRX gene can be, but is not limited to, one or more selected from the group consisting of: a gene encoding human CRX (e.g., NCBI Accession No. NP_000545), such as NCBI Accession No. CRX gene represented by NM_000554 and the like.
  • the retinal function-forming gene may be derived from mammals including primates such as humans and monkeys, rodents such as rats and mice, and the like.
  • the genetic information can be obtained from a known database such as GenBank of National Center for Biotechnology Information (NCBI).
  • the guide nucleic acid is selected from the group consisting of RPE65 gene, GUCY2D gene, SPATA7 gene, AIPL1 gene, LCA5 gene, RPGRIP1 gene, CRB1 gene, CEP290 gene, IMPDH1 gene, RD3 gene, Or a gRNA that binds complementarily to the target sequence of the CRX gene.
  • a " target sequence &quot is a nucleotide sequence present in a target gene or nucleic acid, specifically a nucleotide sequence of a target region or a portion of a target region in a nucleic acid, wherein the " target region " It is a site that can be deformed.
  • target sequence can be used to denote both nucleotide sequence information.
  • the target sequence may be sequence information of a transcribed strand of the target gene DNA, or may be a nucleotide sequence information of a non-transcribed strand.
  • the target sequence may mean 5'-ATCATTGGCAGACTAGTTCG-3 ', which is a transcribed strand of some of the target regions of the target gene A, and a complementary nucleotide sequence (non-transcribed strand) CGAACTAGTCTGCCAATGAT-3 '.
  • the target sequence may be from 5 to 50 nucleotide sequences.
  • the target sequence comprises a sequence of 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Nucleotide sequence or 25 nucleotide sequences.
  • the target sequence includes a guide nucleic acid binding sequence or a guide nucleic acid non-binding sequence.
  • the " guide nucleic acid binding sequence " is a nucleotide sequence having partial or complete complementarity with the guide sequence contained in the guide domain of the guide nucleic acid, and is capable of a complementary binding to a guide sequence contained in the guide domain of the guide nucleic acid.
  • the target sequence and the guide nucleic acid binding sequence can be variously designed according to the target gene or nucleic acid, that is, the nucleotide sequence which can be changed depending on the subject to be genetically modified or corrected.
  • the " guide nucleic acid non-binding sequence &quot is a nucleotide sequence having partial or complete homology with the guide sequence contained in the guide domain of the guide nucleic acid, and can not be complementary to the guide sequence contained in the guide domain of the guide nucleic acid.
  • the guide nucleic acid non-binding sequence is a nucleotide sequence complementary to the guide nucleic acid binding sequence, and can make a complementary binding with the guide nucleic acid binding sequence.
  • the guide nucleic acid binding sequence may be a nucleotide sequence of some of the target sequences and a nucleotide sequence having two different sequence sequences of the target sequence, i.e., one nucleotide sequence of two nucleotide sequences capable of complementary binding to each other .
  • the guide nucleic acid non-binding sequence may be the nucleotide sequence excluding the guide nucleic acid binding sequence in the target sequence.
  • 5'-ATCATTGGCAGACTAGTTCG-3 ' which is the nucleotide sequence of some of the target regions of the target gene A
  • 5'-CGAACTAGTCTGCCAATGAT-3' the complementary nucleotide sequence thereof
  • One of the target sequences i.e., 5'-ATCATTGGCAGACTAGTTCG-3 'or 5'-CGAACTAGTCTGCCAATGAT-3'.
  • the guide nucleic acid non-binding sequence may be 5'-CGAACTAGTCTGCCAATGAT-3 'when the guide nucleic acid binding sequence is 5'-ATCATTGGCAGACTAGTTCG-3', or 5'-CGAACTAGTCTGCCAATGAT-3 '
  • the guide nucleic acid non-binding sequence may be 5'-ATCATTGGCAGACTAGTTCG-3 '.
  • the guide nucleic acid binding sequence may be a target sequence, i.e., a nucleotide sequence identical to the transcribed strand and a selected nucleotide sequence selected from the same nucleotide sequence as the non-transcribed strand.
  • the guide nucleic acid non-binding sequence may be a nucleotide sequence other than the guide nucleic acid binding sequence in the target sequence, i.e., a nucleotide sequence identical to the transcribed strand and a nucleotide sequence selected from the same nucleotide sequence as the non-transcribed strand.
  • the guide nucleic acid binding sequence may be the same as the length of the target sequence.
  • the guide nucleic acid non-binding sequence may be the same as the length of the target sequence or the guide nucleic acid binding sequence.
  • the guide nucleic acid binding sequence may be from 5 to 50 nucleotide sequences.
  • the guide nucleic acid binding sequence comprises 16 nucleotide sequences, 17 nucleotide sequences, 18 nucleotide sequences, 19 nucleotide sequences, 20 nucleotide sequences, 21 nucleotide sequences, 22 nucleotide sequences, 23 nucleotide sequences, 24 nucleotide sequences or 25 nucleotide sequences.
  • the guide nucleic acid non-binding sequence may be from 5 to 50 nucleotide sequences.
  • the guide nucleic acid non-binding sequence comprises 16 nucleotide sequences, 17 nucleotide sequences, 18 nucleotide sequences, 19 nucleotide sequences, 20 nucleotide sequences, 21 nucleotide sequences, 22 nucleotide sequences, 23 nucleotide sequences , 24 nucleotide sequences, or 25 nucleotide sequences.
  • the guide nucleic acid binding sequence may partially or completely complement the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid binding sequence may be the same as the length of the guide sequence.
  • the guide nucleic acid binding sequence may be a nucleotide sequence complementary to a guiding sequence contained in the guiding domain of the guide nucleic acid, and may be at least 70%, 75%, 80%, 85%, 90% or 95% complementary Or a completely complementary nucleotide sequence.
  • the guide nucleic acid binding sequence may or may not include one to eight nucleotide sequences that are not complementary to the guiding sequences contained in the guide domain of the guide nucleic acid.
  • the guide nucleic acid non-binding sequence may have some or complete homology with the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid non-binding sequence may be the same as the length of the guide sequence.
  • the guide nucleic acid non-binding sequence may be a nucleotide sequence having homology to a guide sequence contained in the guide domain of the guide nucleic acid, and may be at least 70%, 75%, 80%, 85%, 90% or 95% Homologous, or completely homologous nucleotide sequence.
  • the guide nucleic acid non-binding sequence may or may not include 1 to 8 nucleotide sequences that are not homologous to the guide sequence contained in the guide domain of the guide nucleic acid.
  • the guide nucleic acid non-binding sequence may be complementary to the guide nucleic acid binding sequence, and the guide nucleic acid non-binding sequence may be the same as the length of the guide nucleic acid binding sequence.
  • the guide nucleic acid non-binding sequence may be a nucleotide sequence complementary to a guide nucleic acid binding sequence, for example at least 90% or 95% or more complementary or completely complementary nucleotide sequence.
  • the guide nucleic acid non-binding sequence may or may not include one or two nucleotide sequences that are not complementary to the guide nucleic acid binding sequence.
  • the guide nucleic acid binding sequence may be a nucleotide sequence located at a position close to the nucleotide sequence recognizable by the editor protein.
  • the guide nucleic acid binding sequence may be a contiguous 5 to 50 nucleotide sequence located adjacent to the 5 'end and / or the 3' end of the nucleotide sequence recognizable by the editor protein.
  • the guide nucleic acid non-binding sequence may be a nucleotide sequence at a position adjacent to the nucleotide sequence recognizable by the editor protein.
  • the guide nucleic acid non-binding sequence may be a contiguous 5 to 50 nucleotide sequence located adjacent to the 5 'end or / and the 3' end of the nucleotide sequence recognizable by the editor protein.
  • &Quot means a complementary binding to a guide nucleic acid binding sequence in a target sequence present in a target gene or nucleic acid.
  • the complementary bond may be 100% complete complementarity, or may be 70% to less than 100% incomplete complementary bond.
  • " targeting gRNA &quot refers to a gRNA that undergoes a complementary binding to a target nucleic acid or a target nucleic acid binding sequence in a target sequence present in the nucleic acid.
  • the target gene disclosed herein may be a retinal function-forming gene.
  • the target genes disclosed herein may be RPE65 gene, GUCY2D gene, SPATA7 gene, AIPL1 gene, LCA5 gene, RPGRIP1 gene, CRB1 gene, CEP290 gene, IMPDH1 gene, RD3 gene, RDH12 gene and / or CRX gene.
  • the target sequence disclosed herein may be a sequence of 10 to 35 contiguous nucleotides located in the promoter region of the retinal function forming gene.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the RPE65 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the GUCY2D gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the SPATA7 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the AIPL1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the LCA5 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the RPGRIP1 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the promoter region of the CRB1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the CEP290 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the IMPDH1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the RD3 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter region of the RDH12 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the promoter region of the CRX gene.
  • the target sequence disclosed herein may be a sequence of 10 to 35 contiguous nucleotides located in the intron region of the retinal function forming gene.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the RPE65 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the GUCY2D gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the SPATA7 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the intron region of the AIPL1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the LCA5 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the RPGRIP1 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the intron region of the CRB1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the CEP290 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the IMPDH1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the RD3 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the intron region of the RDH12 gene.
  • the target sequence may be a contiguous 10 to 25 nucleotide sequence located in the intron region of the CRX gene.
  • the target sequences disclosed herein may be consecutive 10 to 35 nucleotide sequences located in the exon region of the retinal function forming gene.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the RPE65 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the GUCY2D gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the SPATA7 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the AIPL1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the LCA5 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the RPGRIP1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the CRB1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the CEP290 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the IMPDH1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the RD3 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon region of the RDH12 gene.
  • the target sequence may be a contiguous 10 to 25 nucleotide sequence located in the exon region of the CRX gene.
  • the target sequence disclosed herein may be a consecutive 10 to 35 nucleotide sequence located in the enhancer region of the retinal function forming gene.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the enhancer region of the RPE65 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the enhancer region of the GUCY2D gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the enhancer region of the SPATA7 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the enhancer region of the AIPL1 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the enhancer region of the LCA5 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the enhancer region of the RPGRIP1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the enhancer region of the CRB1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the enhancer region of the CEP290 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the enhancer region of the IMPDH1 gene.
  • the target sequence may be a contiguous 10 to 25 nucleotide sequence located in the enhancer region of the RD3 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the enhancer region of the RDH12 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the enhancer region of the CRX gene.
  • Target sequences disclosed herein may be consecutive 10 to 35 nucleotide sequences located in the coding, non-coding, or mixed portion of the retinal function forming gene.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a contiguous 10 to 25 nucleotide sequence located in the encoded, unencrypted, or mixed portion of the RPE65 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the coding, non-coding, or mixed portion of the GUCY2D gene.
  • the target sequence may be a consecutive 10 to 25 nucleotide sequence located in the coding, non-coding, or mixed portion of the SPATA7 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the coding, non-coding, or mixed portion of the AIPL1 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the coding, unencrypted, or mixed portion of the LCA5 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the coding, non-coding, or mixed portion of the RPGRIP1 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the coding, non-coding or mixed portion of the CRB1 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the coding, non-coding, or mixed portion of the CEP290 gene.
  • the target sequence may be a sequence of 10-25 consecutive nucleotides located in the coding, non-coding, or mixed portion of the IMPDH1 gene.
  • the target sequence may be a contiguous 10 to 25 nucleotide sequence located in the coding, non-coding, or mixed portion of the RD3 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the coding, non-coding, or mixed portion of the RDH12 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the coding, non-coding, or mixed portion of the CRX gene.
  • the target sequence disclosed herein may be a contiguous 10 to 35 nucleotide sequence located in the promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof of the retinal function forming gene.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof of the RPE65 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof of the GUCY2D gene.
  • the target sequence may be a consecutive 10-25 nucleotide sequence located in the SPATA7 gene promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof.
  • the target sequence may be a consecutive 10-25 nucleotide sequence located in the AIPL1 gene promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter, enhancer, 3'UTR, polyadenyl (polyA) or a mixed portion thereof of the LCA5 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof of the RPGRIP1 gene.
  • the target sequence may be a consecutive 10-25 nucleotide sequence located in the promoter, enhancer, 3'UTR, polyadenyl (polyA) or a mixed portion thereof of the CRB1 gene.
  • the target sequence may be a contiguous 10-25 nucleotide sequence located in the CEP290 gene promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the IMPDH1 gene promoter, enhancer, 3'UTR, polyadenyl (polyA) or a mixed portion thereof.
  • the target sequence may be a consecutive 10-25 nucleotide sequence located in the promoter, enhancer, 3'UTR, polyadenyl (polyA) or a mixed portion thereof of the RD3 gene.
  • the target sequence may be a sequence of 10-25 contiguous nucleotides located in the promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof of the RDH12 gene.
  • the target sequence may be a consecutive 10-25 nucleotide sequence located in the promoter, enhancer, 3'UTR, polyadenyl (polyA), or a mixed portion thereof of the CRX gene.
  • the target sequence disclosed herein may be a consecutive 10 to 35 nucleotide sequence located in the exon of the retinal function forming gene, an intron, or a mixed portion thereof.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon, intron or mixed portion thereof of the RPE65 gene.
  • the target sequence may be a consecutive 10 to 25 nucleotide sequence located in the exon, intron, or a mixed portion thereof of the GUCY2D gene.
  • the target sequence may be a consecutive 10 to 25 nucleotide sequence located in the exon, intron, or mixed portion thereof of the SPATA7 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon, intron, or mixed portion thereof of the AIPL1 gene.
  • the target sequence may be a consecutive 10 to 25 nucleotide sequence located in the exon, intron, or mixed portion thereof of the LCA5 gene.
  • the target sequence may be a contiguous 10 to 25 nucleotide sequence located in the exon, intron, or mixed portion thereof of the RPGRIP1 gene.
  • the target sequence may be a consecutive 10-25 nucleotide sequence located in the exon, intron or mixed portion of the CRB1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon, intron, or mixed portion thereof of the CEP290 gene.
  • the target sequence may be a consecutive 10 to 25 nucleotide sequence located in the exon, intron, or mixed portion thereof of the IMPDH1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides located in the exon, intron, or mixed portion thereof of the RD3 gene.
  • the target sequence may be a consecutive 10 to 25 nucleotide sequence located in the exon, intron, or mixed portion thereof of the RDH12 gene.
  • the target sequence may be a consecutive 10-25 nucleotide sequence located in the exon, intron, or mixed portion thereof of the CRX gene.
  • a target sequence disclosed herein may comprise a contiguous 10 to 35 nucleotide sequence that comprises or is contiguous with a mutated portion of a retinal function forming gene (e. G., A different portion from a wild-type gene).
  • the mutated portion may be located in the promoter region, the handheld region, the exon region and / or the intron region of the retinal function forming gene.
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the RPE65 gene (e. G., A different portion from the wild type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the GUCY2D gene (e. G., A different portion from the wild-type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the SPATA7 gene (e. G., A different portion from the wild type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the AIPL1 gene (e. G., A different portion from the wild-type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the LCA5 gene (e. G., A different portion from the wild type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the RPGRIP1 gene (e. G., A different portion from the wild type gene).
  • the target sequence may be a consecutive 10-25 nucleotide sequence comprising or contiguous with a mutated portion of the CRB1 gene (e. G., A different portion from the wild-type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the CEP290 gene (e. G., A different portion from the wild type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the IMPDH1 gene (e. G., A different portion from the wild type gene).
  • the target sequence may comprise a contiguous 10-25 nucleotide sequence comprising or contiguous with the mutated portion of the RD3 gene (e. G., A different portion from the wild-type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or contiguous with the mutated portion of the RDH12 gene (e. G., A different portion from the wild type gene).
  • the target sequence may comprise a contiguous 10 to 25 nucleotide sequence comprising or close to a mutated portion of the CRX gene (e. G., A different portion from the wild type gene).
  • the target sequence disclosed herein may be a sequence of 10 to 35 contiguous nucleotides adjacent to the 5 ' and / or 3 ' ends of a proto-spacer-adjacent Motif (PAM) sequence in the nucleic acid sequence of the retinal function forming gene .
  • PAM proto-spacer-adjacent Motif
  • a " proto-spacer-adjacent motif (PAM) sequence " is a nucleotide sequence that is recognized by the editor protein.
  • the PAM sequence may differ in the nucleotide sequence depending on the kind of the editor protein and the species derived.
  • the PAM sequence may be, for example, one or more of the following sequences (described in the 5 'to 3' direction).
  • NGG (wherein N is A, T, C or G);
  • N is independently A, T, C or G, R is A or G, and Y is C or T;
  • N is each independently A, T, C or G and W is A or T;
  • NNNNGATT where each N is independently A, T, C or G;
  • NNGRR (T), where each N is independently A, T, C or G and R is A or G;
  • TTN (where N is A, T, C or G).
  • the target sequence may be a sequence of 10 to 35 nucleotides, a sequence of 15 to 35 nucleotides, a sequence of 20 to 35 nucleotides, a sequence of 25 to 35 nucleotides, or a nucleotide sequence of 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, or 30 to 35 nucleotides.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent to the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the RPE65 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent to the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the GUCY2D gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the SPATA7 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the AIPL1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the LCA5 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the RPGRIP1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the CRB1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent to the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the CEP290 gene.
  • the target sequence may be a contiguous 10 to 25 nucleotide sequence adjacent the 5 'end and / or the 3' end of the PAM sequence in the nucleic acid sequence of the IMPDH1 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the RD3 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the RDH12 gene.
  • the target sequence may be a sequence of 10 to 25 contiguous nucleotides adjacent to the 5 ' and / or 3 ' ends of the PAM sequence in the nucleic acid sequence of the CRX gene.
  • target sequences examples of the target sequences that can be used in one embodiment disclosed herein are summarized in Tables 1 and 2, and the target sequences shown in Tables 1 and 2 are guide nucleic acid non-binding sequences, A complementary sequence, i.e., a guide nucleic acid binding sequence, can be predicted.
  • the target names listed in Tables 1 and 2 are Sp for SpCas9 according to the editor protein and Cj for CjCas9.
  • One aspect of the disclosure taught herein relates to genetically engineered compositions for artificially manipulating or correcting retinal function forming genes.
  • the gene manipulating composition may be used for the production of artificially engineered or calibrated retinal function forming genes.
  • an artificially manipulated or calibrated retinal function-forming gene by the gene-manipulating composition can modulate the retinal function-forming system.
  • artificially modified or engineered or artificially engineered means an artificially deformed state, not a state as it exists in nature.
  • an unnatural artificially manipulated or modified retinal function-forming gene can be used in combination with the term artificial retinal function-forming gene.
  • &quot artificially corrected &quot
  • the correction may be a modification of a normal sequence of a wild-type gene corresponding to a mutation sequence of a gene generated in a natural state, or a modification of a normal protein so that expression of the modified protein is mutated.
  • the calibration may be a modification that causes abnormal expression of the protein due to mutation of the gene occurring in the natural state to be normally expressed.
  • retinal function formation system is a term that encompasses all phenomena that affect retinal function formation by alteration of an artificially engineered retinal function-forming gene, which is directly or indirectly involved in this retinal function formation system Compositions, methods, and uses for which the present invention is directed.
  • retinal function formation regulatory element Each element constituting such a retinal function formation system is collectively referred to as “ retinal function formation regulatory element ".
  • the genetic engineering compositions disclosed herein may comprise a guide nucleic acid and an editor protein.
  • the genetic engineering composition may comprise a guide nucleic acid-editor protein complex.
  • Guided nucleic acid-editor protein complex &quot refers to a complex formed through the interaction of a guide nucleic acid and an editor protein.
  • the " editor protein " means a peptide, polypeptide, or protein that is capable of directly binding to, or not interacting directly with, a nucleic acid.
  • the nucleic acid may be a nucleic acid contained in a target nucleic acid, a gene, or a chromosome.
  • the nucleic acid may be a guide nucleic acid.
  • the editor protein may be an enzyme.
  • &quot enzyme " means a polypeptide or protein comprising a domain capable of cleaving a nucleic acid, gene or chromosome.
  • the enzyme may be a nuclease or a restriction enzyme.
  • the editor protein may comprise a fully active enzyme.
  • the " fully active enzyme &quot means an enzyme having the same function as the original nucleic acid, gene or chromosomal cleavage function of a wild type enzyme.
  • a wild-type enzyme that cleaves a double strand of DNA can be a fully active enzyme that cleaves both double strands of DNA.
  • an artificially engineered enzyme mutant is produced in the same manner as the wild type enzyme If the double strand of DNA is cut, the artificially engineered enzyme variant may be a fully active enzyme.
  • the fully-active enzyme may include an enzyme having an enhanced function than the function of the wild-type enzyme.
  • a particular modified or engineered form of the wild-type enzyme that cleaves the double strand of DNA may have an increased total enzyme activity, i. E. Activity to cleave the increased DNA double strand, over the wild-type enzyme.
  • the editor protein may comprise an incomplete or partially active enzyme.
  • incomplete or partially active enzyme means an enzyme having only a part of the original nucleic acid, gene or chromosome cleavage function of the wild-type enzyme.
  • a particular modified or engineered form of a wild-type enzyme that cleaves a double strand of DNA may be a form having a first function or a form having a second function.
  • the first function is a function of cutting the first strand of the double strand of the DNA
  • the second function is the function of cutting the second strand of the double strand of the DNA.
  • the enzyme having the first function or the enzyme having the second function may be an incomplete or partially activated enzyme.
  • the above-mentioned editor protein may contain an inactive enzyme.
  • inert enzyme means an enzyme in which the original nucleic acid, gene or chromosomal cleavage function of the wild type enzyme is inactivated.
  • a particular modified or engineered form of the wild-type enzyme may be a form in which both the first function and the second function are lost, that is, a first function of cutting the first strand of the double strand of DNA and a second function of cutting the second strand 2 function may be lost.
  • the enzyme in which both the first function and the second function are lost may be an inert enzyme.
  • the above-mentioned editor protein may be a fusion protein.
  • &quot means a protein produced by fusing an additional domain, peptide, polypeptide or protein to an enzyme.
  • the additional domains, peptides, polypeptides or proteins may be functional domains, peptides, polypeptides or proteins having the same or different functions as the functional domains, peptides, polypeptides or proteins contained in the enzyme.
  • the fusion protein is at or near the amino terminus of the enzyme; Carboxy terminus or vicinal thereof; The middle part of the enzyme; Or a combination of these functional domains, peptides, polypeptides or proteins in one or more of these combinations.
  • the functional domain, the peptide, the polypeptide or the protein may be selected from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription activity, peptides, polypeptides, or proteins that have a release factor activity, a histone modification activity, a cleavage activity or a nucleic acid binding activity, or a separation of proteins (including peptides) But is not limited to, a tag or reporter gene for purification.
  • the functional domains, peptides, polypeptides or proteins may be deaminases.
  • the tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag, (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • beta-glucuronidase luciferase
  • green fluorescent protein but are not limited to, autofluorescent proteins, including, for example, GFP), HcRed, DsRed, Cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP
  • the functional domain, peptide, polypeptide or protein may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).
  • NLS nuclear localization sequence or signal
  • NES nuclear export sequence or signal
  • the NLS is an NLS of the SV40 virus large T-antigen with the amino acid sequence PKKKRKV; NLS from nucleoplasmin (e. G., Nucleoplasmin NLS with sequence KRPAATKKAGQAKKKK); C-myc NLS with amino acid sequence PAAKRVKLD or RQRRNELKRSP; HRNPA1 M9 NLS with sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; The sequence of the IBB domain from IMPOTIN-ALPHA RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; Sequences of myoma T protein VSRKRPRP and PPKKARED; Sequence of human p53 POPKKKPL; Sequence of mouse c-abl IV SALIKKKKKMAP; The sequences DRLRR and PKQKKRK of influenza virus NS1; The sequence of the hepatitis virus delta antigen RKLKKKIKKL
  • the additional domains, peptides, polypeptides or proteins may be non-functional domains, peptides, polypeptides or proteins that do not perform a particular function.
  • the non-functional domains, peptides, polypeptides or proteins may be domains, peptides, polypeptides or proteins that do not affect the function of the enzyme.
  • the fusion protein is at or near the amino terminus of the enzyme; Carboxy terminus or vicinal thereof; The middle part of the enzyme; Or a combination of one or more of these non-functional domains, peptides, polypeptides or proteins.
  • the above-mentioned editor protein may be an enzyme or a fusion protein existing in a natural state.
  • the above-mentioned editor protein may be a native form of the enzyme or a modified form of a part of the fusion protein.
  • the above-mentioned editor protein may be an artificially generated enzyme or a fusion protein which is not present in a natural state.
  • the above-mentioned editor protein may be an artificially generated enzyme or a part of the fusion protein which is not present in a natural state.
  • the modification may be substitution, deletion, addition, or a mixture of amino acids contained in the editor protein.
  • the modification may be substitution, deletion, addition, or a mixture of some of the nucleotides in the nucleotide sequence encoding the editor protein.
  • the gene manipulating composition may further comprise a donor comprising a specific nucleotide sequence to be inserted, or a nucleic acid sequence encoding the donor.
  • the insertion may be a nucleotide sequence of a desired nucleotide sequence of a retinal function-forming gene.
  • the insertion may be a nucleic acid sequence for correcting a mutation of a retinal function-forming gene to be manipulated or introducing a mutation.
  • the " donor” refers to a nucleotide sequence that aids in repair by homologous recombination of a damaged gene or nucleic acid.
  • the donor may be a double-stranded nucleic acid or a single-stranded nucleic acid.
  • the donor may be linear or circular.
  • the donor may comprise a nucleotide sequence that is homologous to the target gene or nucleic acid.
  • the donor may comprise a nucleotide sequence that is homologous to a nucleotide sequence on the upstream and downstream of a damaged nucleic acid, respectively, at a position where a particular nucleotide sequence is desired to be inserted.
  • the particular nucleotide sequence to be inserted may be located between a nucleotide sequence that is homologous to the right nucleotide sequence of the damaged nucleic acid and a nucleotide sequence that is homologous to the left nucleotide sequence of the damaged nucleic acid.
  • the homologous nucleotide sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% You can have same sex.
  • the donor may comprise a particular nucleic acid sequence.
  • the specific nucleic acid sequence may be a partial nucleotide sequence of the target gene or some similar nucleotide.
  • Some nucleotide sequences of the target gene may include normal nucleic acid sequences that have been mutagenized to correct, for example, a target gene comprising a mutation.
  • some of the pseudonucleotide sequences of the target gene may include a mutation-inducing nucleic acid sequence in which some of the normal nucleic acid sequences of the target gene are mutated to mutate the normal target gene.
  • the specific nucleic acid when a gene having a mutation including a termination codon in the exon is used as a target gene, the specific nucleic acid may be a normal nucleotide sequence capable of correcting a termination codon in the exon to a normal nucleotide sequence.
  • the specific nucleic acid when the expression of the target gene is to be suppressed, the specific nucleic acid may be a termination codon to be inserted into the exon of the target gene. In this case, The expression of the target gene can be inhibited by the inserted termination codon in the exon.
  • the specific nucleic acid sequence may be a termination codon.
  • the specific nucleic acid sequence may be an exogenous nucleic acid sequence.
  • the exogenous nucleic acid sequence may be an exogenous gene that is desired to be expressed in a cell containing the target gene.
  • the specific nucleic acid sequence may be a nucleic acid sequence desired to be expressed in a cell containing the target gene.
  • the nucleic acid sequence may be a specific gene expressed in a cell containing a target gene.
  • the specific gene is increased in the number of intracellular copies by the gene-acting composition comprising the donor .
  • the donor may optionally comprise an additional nucleotide sequence.
  • the ancillary nucleic acid sequence may serve to enhance donor stability, target insertion efficiency, or homologous recombination efficiency.
  • the ancillary nucleotide sequence may be a nucleic acid sequence rich in nucleotides A, T, i.e., an A-T rich domain.
  • the ancillary nucleotide sequence may be a scaffold / matrix attachment region (SMAR).
  • the guide nucleic acid, the editor protein, or the guide nucleic acid-editor protein complex disclosed herein can be delivered or introduced into the subject in various forms.
  • the subject may be an organism comprising the target gene or chromosome of the guide nucleic acid-editor protein complex.
  • the organism may be an animal, an animal tissue, or an animal cell.
  • the organism may be a human, a human tissue or a human cell.
  • the tissue may be an eye, skin, liver, kidney, heart, lung, brain, muscle or blood.
  • the cells may be retinal cells, nerve cells, glial cells, cone cells, glandular cells, retinal pigment epithelial cells or stem cells.
  • the sample or the sample is obtained from an organism including a target gene or a chromosome such as a needle, blood, retinal tissue, brain tissue, retinal cell, nerve cell, glial cell, cone cell, glandular cell, retinal pigment epithelial cell or stem cell Lt; / RTI >
  • a target gene or a chromosome such as a needle, blood, retinal tissue, brain tissue, retinal cell, nerve cell, glial cell, cone cell, glandular cell, retinal pigment epithelial cell or stem cell Lt; / RTI >
  • the subject may be an organism comprising a retinal function-forming gene.
  • the guide nucleic acid, the editor protein, or the guide nucleic acid-editor protein complex may be delivered or introduced into the subject in the form of DNA, RNA, or a mixture thereof.
  • the form of DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or the editor protein can be transferred or introduced into the subject by methods known in the art.
  • the form of DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject by a vector, non-vector, or a combination thereof.
  • the vector may be a viral or non-viral vector (e. G., A plasmid).
  • the non-vector may be naked DNA, DNA complex or mRNA.
  • the nucleic acid sequence encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject by a vector.
  • the vector may comprise a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein.
  • the vector may comprise a guide nucleic acid and a nucleic acid sequence encoding the editor protein at the same time.
  • the vector may comprise a nucleic acid sequence encoding a guide nucleic acid.
  • the domains included in the guide nucleic acid may be included in one vector or may be divided into individual vectors.
  • the vector may comprise a nucleic acid sequence encoding an editor protein.
  • the nucleic acid sequence encoding the editor protein may be contained in one vector, or the nucleic acid sequence encoding the editor protein may be divided into several vectors.
  • the vector may comprise one or more control / control components.
  • the regulatory / control component may include a capture motor, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor, and / or a 2A Sequence.
  • a capture motor an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor, and / or a 2A Sequence.
  • the promoter may be a promoter recognized by RNA polymerase II.
  • the promoter may be a promoter recognized by RNA polymerase III.
  • the promoter may be an inducible promoter.
  • the promoter may be a target specific promoter.
  • the promoter may be a virus or a non-viral promoter.
  • the promoter may use a suitable promoter depending on the control region (i.e., the nucleic acid sequence encoding the guide nucleic acid or the editor protein).
  • a promoter useful for the guide nucleic acid may be a H1, EF-1a, tRNA or U6 promoter.
  • the promoter useful for the editor protein may be CMV, EF-1a, EFS, MSCV, PGK or CAG promoter.
  • the vector may be a viral vector or a recombinant viral vector.
  • the virus may be a DNA virus or an RNA virus.
  • the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.
  • dsDNA double-stranded DNA
  • ssDNA single-stranded DNA
  • the RNA virus may be a single stranded RNA (ssRNA) virus.
  • ssRNA single stranded RNA
  • the virus may be, but is not limited to, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus or herpes simplex virus.
  • the virus can infect a host (e. G., A cell), introduce a nucleic acid encoding the viral genetic information into the host, or insert a nucleic acid encoding the genetic information into the host's genome.
  • a host e. G., A cell
  • the virus having these characteristics can be used to introduce the guide nucleic acid and / or the editor protein into the subject.
  • the guide nucleic acid and / or the editor protein introduced using the virus can be transiently expressed in a subject (e.g., a cell).
  • a guide nucleic acid and / or an editor protein introduced using a virus can be detected in a subject (for example, a cell) for a long period of time (for example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months , 9 months, 1 year, 2 years or permanently).
  • the packaging capacity of the virus is at least 2kb to 50kb and may vary depending on the virus type. According to this packaging ability, it is possible to design a viral vector containing a guide nucleic acid or an editor protein singly or to design a viral vector containing both a guide nucleic acid and an editor protein. Or a viral vector containing the guide nucleic acid, the editor protein, and additional components.
  • the nucleic acid sequence encoding the guide nucleic acid and / or the editor protein can be delivered or introduced by the recombinant lentivirus.
  • a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein may be delivered or introduced by a recombinant adenovirus.
  • nucleic acid sequence encoding the guide nucleic acid and / or the editor protein may be delivered or introduced by recombinant AAV.
  • nucleic acid sequence encoding the guide nucleic acid and / or the editor protein may be delivered or introduced by a hybrid of at least one of the hybrid viruses, e. G., The viruses described herein.
  • nucleic acid sequence encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject as a non-vector.
  • the non-vector may comprise a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein.
  • the non-vector may be naked DNA, DNA complex, mRNA or a mixture thereof.
  • the non-vector may be selected from the group consisting of electroporation, gene gun, ultrasonication, magnetofection, transient cell compaction or squeezing (see, for example, Lee, et al, (2012) Nano Lett., 12, 6322- 6327), lipid-mediated transfection, dendrimers, nanoparticles, calcium phosphate, silica, silicates (orthosilicates) or a combination thereof.
  • delivery via electroporation can be performed by mixing the cell with a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein in a cartridge, chamber, or cuvette and applying electrical stimulation of defined duration and amplitude have.
  • non-vectors can be delivered using nanoparticles.
  • the nanoparticles may be inorganic nanoparticles (e.g., magnetic nanoparticles, silica, etc.) or organic nanoparticles (e.g., lipids coated with polyethylene glycol (PEG)).
  • the outer surface of the nanoparticles may be conjugated with a positively charged polymer (e. G., Polyethyleneimine, polylysine, polyserine, etc.) that allows attachment.
  • a positively charged polymer e. G., Polyethyleneimine, polylysine, polyserine, etc.
  • it can be delivered using a lipid envelope.
  • Exosomes are endogenous nano-vesicles that transport proteins and RNA that can deliver RNA to the brain and other target organs.
  • the liposomes are spherical structures composed of a single or multiple lamellar lipid bilayer surrounding the internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; Phospholipids are most commonly used to produce liposomes as drug carriers.
  • composition for delivery of the non-vector may comprise several other additives.
  • the editor protein can be delivered or introduced into a subject in the form of a peptide, polypeptide or protein.
  • the form of the peptide protein, polypeptide or protein can be delivered or introduced into the subject by methods known in the art.
  • the form of the peptide, polypeptide or protein can be determined by electroporation, microinjection, transient cell compaction or squeezing (see, for example, Lee, et al, (2012) Nano Lett., 12, 6322-6327) , Lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery, or a combination thereof.
  • the delivery of the peptide, polypeptide or protein may be delivered with a nucleic acid sequence encoding a guide nucleic acid.
  • delivery via electroporation can be performed by mixing the cells with a guide nucleic acid in a cartridge, chamber, or cuvette, or with a cell to introduce the editor protein without a guide nucleic acid, and applying an electrical stimulus of defined duration and amplitude .
  • the guide nucleic acid and the editor protein can be delivered or introduced into the subject in the form of a nucleic acid-protein mixture.
  • the guide nucleic acid and the editor protein may be transferred or introduced into the subject in the form of a guide nucleic acid-editor protein complex.
  • the guide nucleic acid may be DNA, RNA, or a mixture thereof.
  • the editor protein may be in the form of a peptide, polypeptide or protein.
  • the guide nucleic acid and the editor protein can be delivered or introduced into the subject in the form of a guide nucleic acid-editor protein complex, that is, a ribonucleoprotein (RNP), in the form of an RNA in the form of a guide nucleic acid and a protein in the form of a protein.
  • a guide nucleic acid-editor protein complex that is, a ribonucleoprotein (RNP)
  • RNP ribonucleoprotein
  • the guide nucleic acid-editor protein complexes disclosed herein may modify a target nucleic acid, gene or chromosome.
  • a guide nucleic acid-editor protein complex induces modifications to the sequence of a target nucleic acid, gene or chromosome.
  • the protein expressed by the target nucleic acid, gene or chromosome can be modified in structure and / or function, the expression of the protein can be regulated, or the expression of the protein can be eliminated.
  • the guide nucleic acid-editor protein complex can act at the DNA, RNA, gene or chromosome level.
  • the guide nucleic acid-editor protein complex may manipulate or modify a target gene to control (e.g., suppress, inhibit, decrease, increase, or promote) expression of a protein encoded by the target gene, For example, inhibiting, inhibiting, reducing, increasing or promoting) or a modified protein.
  • the guide nucleic acid-editor protein complex can act at the transcription and translation step of the gene.
  • the guide nucleic acid-editor protein complex may modulate (e.g., inhibit, inhibit, decrease, increase, or promote) the expression of a protein encoded by the target gene by promoting or inhibiting transcription of the target gene.
  • the guide nucleic acid-editor protein complex can modulate (e.g., inhibit, inhibit, reduce, increase, or promote) the expression of a protein encoded by the target gene by promoting or inhibiting translation of the target gene.
  • the genetic engineering composition may comprise a gRNA and a CRISPR enzyme.
  • the gene manipulating composition may comprise a gRNA-CRISPR enzyme complex.
  • gRNA-CRISPR enzyme complex &quot means a complex formed through interaction of gRNA with CRISPR enzyme.
  • the gRNA related description is as described above.
  • the " CRISPR enzyme " is a major protein component of the CRISPR-Cas system and forms a complex with gRNA to form the CRISPR-Cas system.
  • the CRISPR enzyme may be a nucleic acid or a polypeptide (or protein) having a sequence encoding a CRISPR enzyme.
  • the CRISPR enzyme may be a Type II CRISPR enzyme.
  • the crystal structure of the Type II CRISPR enzyme is similar to that of two or more naturally occurring microorganisms Type II CRISPR enzyme molecules (Jinek et al., Science, 343 (6176): 1247997, 2014), and a complex of Streptococcus pyogenes (Nishimasu et al., Cell, 156: 935-949, 2014; and Anders et al., Nature, 2014, doi: 10.1038 / nature13579).
  • Type II CRISPR enzymes include two lobes, Recognition (REC) and Nucleic Acid (NUC) lobes, each lobe containing multiple domains.
  • REC Recognition
  • NUC Nucleic Acid
  • the REC lobe comprises an arginine-rich bridge spiral (BH), a REC1 domain and a REC2 domain.
  • BH arginine-rich bridge spiral
  • the BH domain is a long? -Helical and arginine rich region, and the REC1 and REC2 domains play an important role in the recognition of the double strand formed in the gRNA, for example, single strand gRNA, double gRNA or tracrRNA.
  • the NUC lobe comprises the RuvC domain, the HNH domain and the PAM-interacting (PI) domain.
  • the RuvC domain is used to mean the RuvC-like domain
  • the HNH domain is used to mean the HNH-like domain.
  • the RuvC domain shares structural similarity with members of a microorganism existing in a natural state including a Type II CRISPR enzyme, and has a single strand, for example, a complementary strand of a target gene or a nucleic acid, The strands that do not bind are cleaved.
  • the RuvC domain is often referred to in the art as the RuvCI domain, the RuvCII domain, and the RuvCIII domain, typically RuvC I, RuvCII, and RuvCIII.
  • the HNH domain shares structural similarity with the HNH endonuclease and cleaves a single strand, for example, a complementary strand of the target nucleic acid molecule, i. E., A strand that makes a complementary binding to the gRNA.
  • the HNH domain is located between the RuvC II and III motifs.
  • the PI domain recognizes a specific nucleotide sequence in the target gene or nucleic acid, that is, PAM (Protospacer adjacent motif) or interacts with PAM.
  • PAM Protospacer adjacent motif
  • the PAM may be different depending on the origin of the Type II CRISPR enzyme.
  • PAM may be 5'-NNNNGATT-3 'when Naceria meningitidis Cas9 (NmCas9)
  • PAM is determined depending on the origin of the above-mentioned enzymes. However, as the mutation of the enzyme derived from the enzyme is studied, the PAM may be different.
  • the Type II CRISPR enzyme may be Cas9.
  • the Cas9 may be selected from the group consisting of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Campylobacter jejuni ), Nocardiopsis rougei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes (Streptomyces viridochromogenes), Streptomyces viridochromogenes ), Streptosporangium roseum, Streptosporangium roseum, Alicyclobaclus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitii, Bacillus spp.
  • the microorganisms of the present invention are preferably selected from the group consisting of Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, For example, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, , Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldic effetosiruptor bescii, Candidatus de sulphorus (Candida albicans), Candida albicans Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, F inactivia magna, Natranaerobius thermophilus, Pe
  • Spumigena Nostoc sp., Arthrospira maxima, Arthrospira platensis, , Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, ), Thermosipho africanus or Acaryochloris marina, and the like.
  • Cas9 is an enzyme which cleaves a target sequence or position on a target gene or a nucleic acid by binding with gRNA, and is an HNH domain in which a gRNA can cleave a nucleic acid strand that makes a complementary binding, A RuvC domain that is capable of cleaving a nucleic acid strand that carries a nucleic acid strand that is capable of cleaving a nucleic acid strand that carries a target nucleic acid, a target, i.e., a REC domain that interacts with the target, and a PI domain that recognizes PAM. Concrete structural characteristics of Cas9 are described by Hiroshi Nishimasu et al. (2014) Cell 156: 935-949.
  • the Cas9 may be isolated from a microorganism existing in a natural state or produced naturally by a recombinant method or a synthetic method.
  • the CRISPR enzyme may be a Type V CRISPR enzyme.
  • the Type V CRISPR enzyme has a similar RuvC domain corresponding to the RuvC domain of the Type II CRISPR enzyme and lacks the HNH domain of the Type II CRISPR enzyme and instead contains the Nuc domain and the REC domain interacting with the target and the WED Domain and a PI domain that recognizes PAM.
  • the structural characteristics of the specific Type V CRISPR enzyme are described in Takashi Yamano et al. (2016) Cell 165: 949-962.
  • the Type V CRISPR enzyme can interact with the gRNA and form a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, and cooperate with the gRNA to approximate the guiding sequence with the target sequence containing the PAM sequence.
  • a CRISPR complex i.e., a CRISPR complex
  • the ability of the Type V CRISPR enzyme to interact with the target gene or nucleic acid depends on the PAM sequence.
  • the PAM sequence can be recognized by the PI domain of Type V CRISPR enzyme, which is a sequence existing in the target gene or nucleic acid.
  • the sequence of the PAM sequence may be different depending on the origin of the Type V CRISPR enzyme. That is, there is a PAM sequence that can be recognized specifically for each species.
  • the PAM sequence recognized by Cpf1 may be 5'-TTN-3 '(N is A, T, C or G).
  • N is A, T, C or G
  • PAM is determined depending on the origin of the above-mentioned enzymes.
  • the mutation of the enzyme derived from the enzyme is studied, the PAM may be different.
  • the Type V CRISPR enzyme may be Cpf1.
  • the Cpf1 is Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus , Cpf1 from Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus.
  • the Cpf1 has a similar RuvC domain corresponding to the RuvC domain of Cas9, lacks the HNH domain of Cas9, contains the Nuc domain instead, and has a REC domain and a WED domain interacting with the target and a PI domain .
  • Concrete structural features of Cpf1 are described by Takashi Yamano et al. (2016) Cell 165: 949-962.
  • the Cpf1 may be isolated from a microorganism existing in a natural state, or produced naturally by a recombinant method or a synthetic method.
  • the CRISPR enzyme may be a nuclease or a restriction enzyme having a function of cleaving a target gene or a double strand of nucleic acid.
  • the CRISPR enzyme may be a fully active CRISPR enzyme.
  • Fully active means a state having the same function as that of a wild-type CRISPR enzyme, and the CRISPR enzyme in this state is referred to as a fully active CRISPR enzyme.
  • the "wild type CRISPR enzyme function” refers to a function of cleaving a double strand of DNA, that is, a first function of cutting a first strand of a double strand of DNA and a second function of cutting a second strand of DNA And a second function of performing the second function.
  • the fully active CRISPR enzyme may be a wild type CRISPR enzyme that cleaves double strands of DNA.
  • the fully active CRISPR enzyme may be a CRISPR enzyme variant that has modified or manipulated a wild type CRISPR enzyme that cleaves double strands of DNA.
  • the CRISPR enzyme mutant may be an enzyme in which at least one amino acid of the amino acid sequence of the wild-type CRISPR enzyme is replaced with another amino acid or at least one amino acid has been removed.
  • the CRISPR enzyme mutant may be an enzyme having one or more amino acids added to the amino acid sequence of wild-type CRISPR enzyme. At this time, the position of the added amino acid may be within the N-terminus, C-terminus or amino acid sequence of the wild-type enzyme.
  • the CRISPR enzyme mutant may be a completely active enzyme having improved function than the wild-type CRISPR enzyme.
  • modified or engineered forms of wild-type CRISPR enzymes i.e., CRISPR enzyme variants
  • CRISPR enzyme variants can not cut DNA double strands that are to be cleaved or can cut DNA double strands while maintaining constant distance spacing.
  • the modified or engineered form may be a fully active CRISPR enzyme with enhanced functional activity over wild-type CRISPR enzymes.
  • the CRISPR enzyme variant may be a fully active CRISPR enzyme with reduced function than the wild type CRISPR enzyme.
  • a particular modified or engineered form of the wild-type CRISPR enzyme can cut double strands of DNA in a state in which the DNA double strand to be cleaved is in close proximity or a specific bond is formed.
  • the specific binding may be, for example, a combination of an amino acid at a specific position of the enzyme and a DNA nucleotide sequence at a cleavage site.
  • the modified or engineered form may be a fully active CRISPR enzyme with reduced functional activity than the wild-type CRISPR enzyme.
  • the CRISPR enzyme may be an incomplete or partially active CRISPR enzyme.
  • Incomplete or partially active &quot refers to a function of the wild-type CRISPR enzyme, i.e., a function selected from among a first function of cleaving a first strand of a double strand of DNA and a second function of severing a second strand of a double strand of DNA It means the state of having.
  • the CRISPR enzyme in this state is termed an incomplete or partially active CRISPR enzyme.
  • the incomplete or partially active CRISPR enzyme may also be referred to as a nickase.
  • the nicarase may have nuclease activity by the RuvC domain of the CRISPR enzyme. That is, the nicarase may not contain nuclease activity by the HNH domain of the CRISPR enzyme, for which the HNH domain may be manipulated or altered.
  • the nicarase may be a Type II CRISPR enzyme comprising a modified HNH domain.
  • the Type II CRISPR enzyme is a wild-type SpCas9
  • the nicarase can mutate histone of amino acid sequence 840 of wild-type SpCas9 to alanine to be an SpCas9 mutant in which the nuclease activity of the HNH domain is inactivated have.
  • the produced niacase, that is, the SpCas9 mutant has nuclease activity by the RuvC domain, so that it can cleave the target gene or the non-complementary strand of the nucleic acid, that is, the strand that does not bind complementary to the gRNA.
  • the Type II CRISPR enzyme is a wild-type CjCas9
  • the nicarase mutates histidine of amino acid sequence 559 of wild-type CjCas9 to alanine to generate a CjCas9 mutant in which the nuclease activity of the HNH domain is inactivated Lt; / RTI >
  • the produced niacase, that is, the CjCas9 mutant has nuclease activity by the RuvC domain, so that it is possible to cleave the target gene or the non-complementary strand of the nucleic acid, that is, the strand which is not complementary to the gRNA.
  • the nicarase may have nuclease activity by the HNH domain of the CRISPR enzyme. That is, the nicacase may not contain nuclease activity by the RuvC domain of the CRISPR enzyme, and the RuvC domain may be manipulated or altered for this purpose.
  • the nicarase may be a Type II CRISPR enzyme comprising a modified RuvC domain.
  • the Type II CRISPR enzyme is wild-type SpCas9
  • the nicacase mutant s the amino acid sequence 10 aspartic acid of wild-type SpCas9 to alanine to generate a SpCas9 mutant in which the nuclease activity of the RuvC domain is inactivated .
  • the produced niacase that is, the SpCas9 mutant has nuclease activity by the HNH domain, so that the complementary strand of the target gene or the nucleic acid, that is, the strand that makes a complementary bond to the gRNA, can be cleaved.
  • the niacase mutates the aspartate acid at amino acid sequence 8 of wild-type CjCas9 to alanine to generate CjCas9 in which the nuclease activity of the RuvC domain is inactivated Lt; / RTI >
  • the produced niacase, that is, the CjCas9 mutant has nuclease activity by the HNH domain, so that the complementary strand of the target gene or the nucleic acid, that is, the strand that makes a complementary bond to the gRNA, can be cleaved.
  • the CRISPR enzyme may be an inactive CRISPR enzyme.
  • Inactive &quot means a state in which both the function of the wild-type CRISPR enzyme, i.e., the first function of cleaving the first strand of the double strand of DNA and the second function of severing the second strand of the double strand of DNA, are lost.
  • the CRISPR enzyme in this state is termed an inactive CRISPR enzyme.
  • the inactive CRISPR enzyme may have nuclease inactivation due to mutation in the domain having nuclease activity of the wild-type CRISPR enzyme.
  • the inactive CRISPR enzyme may have nuclease abilities due to mutations in the RuvC domain and the HNH domain. That is, the inactive CRISPR enzyme may not contain nuclease activity by the RuvC domain and the HNH domain of the CRISPR enzyme, and the RuvC domain and the HNH domain may be manipulated or modified for this purpose.
  • the inactive CRISPR enzyme may be a Type II CRISPR enzyme comprising a modified RuvC domain and an HNH domain.
  • the inactive CRISPR enzyme mutates allantoin 10-aspartic acid and 840-histidine of wild-type SpCas9 into alanine to form a nucleucase of RuvC domain and HNH domain Activity may be an inactivated SpCas9 mutant.
  • the generated inactive CRISPR enzyme that is, the SpCas9 mutant, can not cleave the double strand of the target gene or the nucleic acid because the nuclease activity of the RuvC domain and the HNH domain is inactivated.
  • the generated inactive CRISPR enzyme that is, the CjCas9 mutant, can not cleave the double strand of the target gene or the nucleic acid because the nuclease activity of the RuvC domain and the HNH domain is inactivated.
  • the CRISPR enzyme may have a helicase activity, i.e., the ability to resolve the helical structure of the double-stranded nucleic acid.
  • the CRISPR enzyme can modify the CRISPR enzyme to be fully active, incomplete or partially active, or inert to the helicase activity of the CRISPR enzyme.
  • the CRISPR enzyme may be a CRISPR enzyme variant that artificially manipulates or modifies a wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may be artificially modified to alter the function of the wild-type CRISPR enzyme, i.e., the first function of cleaving the first strand of the double strand of DNA and / or the second function of severing the second strand of the double strand of DNA Manipulated or modified CRISPR enzyme variants.
  • the CRISPR enzyme mutant may be one in which the first function of the wild-type CRISPR enzyme is lost.
  • the CRISPR enzyme mutant may be a form in which the function of the wild-type CRISPR enzyme is lost in the second function.
  • the CRISPR enzyme mutant may be a form in which the function of the wild-type CRISPR enzyme, i.e., the first function and the second function, is lost.
  • the CRISPR enzyme mutant can form a gRNA-CRISPR enzyme complex through interaction with gRNA.
  • the CRISPR enzyme mutant may be an artificially manipulated or modified CRISPR enzyme mutant to alter the function of interacting with the gRNA of the wild-type CRISPR enzyme.
  • the CRISPR enzyme variant may be in a reduced form of interaction with the gRNA relative to the wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may be in an increased form of interaction with the gRNA relative to the wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may have a first function of the wild-type CRISPR enzyme and a reduced interaction with the gRNA.
  • the CRISPR enzyme mutant may have a first function of the wild-type CRISPR enzyme and an increased interaction with the gRNA.
  • the CRISPR enzyme mutant may have a second function of the wild-type CRISPR enzyme and a reduced interaction with the gRNA.
  • the CRISPR enzyme mutant may have a second function of the wild-type CRISPR enzyme and an increased interaction with the gRNA.
  • the CRISPR enzyme mutant may be a form in which the interaction with the gRNA is reduced without having the first function and the second function of the wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may be one in which the interaction with the gRNA is increased without having the first function and the second function of the wild-type CRISPR enzyme.
  • various gRNA-CRISPR enzyme complexes may be formed depending on the intensity of interaction between the gRNA and the CRISPR enzyme mutant, and the function of accessing or cleaving the target sequence may be different depending on the CRISPR enzyme mutant.
  • a gRNA-CRISPR enzyme complex formed by a CRISPR enzyme variant with reduced interaction with a gRNA will only bind double or single strands of the target sequence if it is proximate or localized to a target sequence that is fully complementary to the gRNA Can be cut.
  • the CRISPR enzyme mutant may be a modification of at least one amino acid of the amino acid sequence of the wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may be a substitution of at least one amino acid of the amino acid sequence of the wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may be one in which at least one amino acid of the wild type CRISPR enzyme amino acid sequence has been removed.
  • the CRISPR enzyme mutant may have at least one amino acid added to the amino acid sequence of the wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may be substituted, deleted, and / or added to at least one amino acid in the amino acid sequence of the wild-type CRISPR enzyme.
  • the CRISPR enzyme mutant may be selected from the group consisting of the original function of the wild-type CRISPR enzyme, i.e., the first function of cutting the first strand of the double strand of DNA and the second function of cutting the second strand of DNA, (functional domain). At this time, the CRISPR enzyme mutant may have an additional function in addition to the original function of the wild-type CRISPR enzyme.
  • the functional domain may be selected from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification a cleavage activity or a nucleic acid binding activity or a tag or reporter gene for the separation and purification of a protein (including a peptide) It does not.
  • the tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag, (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • beta-glucuronidase luciferase
  • green fluorescent protein but are not limited to, autophosphor proteins, including GFP, HcRed, DsRed, Cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP).
  • the functional domain may be deaminase.
  • an incomplete or partial CRISPR enzyme may additionally include cytidine deaminase as a functional domain.
  • a fusion protein can be generated by adding SpCas9 nicarase stanidine diamine, for example APOBEC1 (apolipoprotein B editing complex 1).
  • SpCas9 nicarase stanidine diamine for example APOBEC1 (apolipoprotein B editing complex 1).
  • APOBEC1 apolipoprotein B editing complex 1
  • the thus formed [SpCas9 niacase] - [APOBEC1] can be used for nucleotide C or T nucleotide correction or editing, or nucleotide G nucleotide A nucleotide correction or editing.
  • an incomplete or partial CRISPR enzyme may further include adenine deaminase as a functional domain.
  • a fusion protein can be generated by adding adenine diamines to the SpCas9 nicarase, such as TadA variants, ADAR2 variants, ADAT2 variants, and the like.
  • nucleotide A transforms nucleotide A into inosine, Since nucleotide A is recognized as nucleotide G and substantially nucleotide A is modified or edited as G nucleotide, nucleotide A can be used for G nucleotide correction or editing, or nucleotide T can be used for nucleotide correction or editing .
  • the functional domain may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).
  • NLS nuclear localization sequence or signal
  • NES nuclear export sequence or signal
  • the CRISPR enzyme may comprise one or more NLSs.
  • the NLS is at or near the amino terminus of the CRISPR enzyme; Carboxy terminus or vicinal thereof; Or a combination thereof.
  • the NLS can be, but is not limited to, the NLS sequence derived from: the NLS of the SV40 virus large T-antigen with the amino acid sequence PKKKRKV; NLS from nucleoplasmin (e.
  • the CRISPR enzyme mutant may include a CRISPR enzyme in a split form divided into two or more parts by dividing the CRISPR enzyme. &Quot; Split " means dividing a protein functionally or structurally or optionally into two or more.
  • the split type CRISPR enzyme may be a fully active enzyme, an incomplete or partially active enzyme, or an inactive enzyme.
  • split SpCas9 can be generated by dividing between 656 tyrosine and 657 threonine into two parts.
  • the split form of the CRISPR enzyme may optionally include additional domains, peptides, polypeptides or proteins for reconstitution.
  • Additional domains, peptide polypeptides or proteins for such reconstitution may be assembled such that the split form of the CRISPR enzyme is structurally identical or similar to the wild-type CRISPR enzyme.
  • Additional domains, peptides, polypeptides or proteins for the reconstitution include FRB and FKBP dimerization domains; Intein; ERT and VPR domains, or domains that form heterodimers in certain conditions.
  • the FRB domain may be connected to one of the two fragments and the FKBP domain may be ligated to the other fragment of the split SpCas9, which is obtained by dividing the fragment between serine 713 and glycine 714 have.
  • Split SpCas9 thus produced can dimerize the FRB domain and the FKBP domain in the presence of rapamycin to generate a reconstituted CRISPR enzyme.
  • the CRISPR enzyme or CRISPR enzyme variant disclosed by the present specification may be a polypeptide, a protein, or a nucleic acid having a sequence encoding the same, wherein the CRISPR enzyme or a CRISPR enzyme variant may be codon-optimized Lt; / RTI >
  • codon optimization &quot is meant the use of at least one codon of the native sequence to replace the nucleic acid of the host cell with a codon that is more frequently or most frequently used, It means a process of transforming the sequence.
  • Various species have specific biases for a particular codon of a particular amino acid and codon bias (difference in codon usage between organisms) is often correlated with the efficiency of translation of the mRNA, which is dependent on the nature of the codon being translated and the availability of a particular tRNA molecule As shown in FIG.
  • the predominance of the selected tRNA in the cell generally reflects the codons most frequently used for peptide synthesis.
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • gRNA, CRISPR enzyme or gRNA-CRISPR enzyme complexes disclosed herein can be delivered or introduced into a subject in a variety of forms.
  • the gRNA and / or CRISPR enzyme can be delivered or introduced into a subject by a vector comprising a nucleic acid sequence encoding each.
  • the vector may comprise a nucleic acid sequence encoding a gRNA and / or a CRISPR enzyme.
  • the vector may simultaneously contain a gRNA and a nucleic acid sequence encoding a CRISPR enzyme.
  • the vector may comprise a gRNA-encoding nucleic acid sequence.
  • the domains contained in the gRNA may be included in one vector, or divided into respective domains and included in each vector.
  • the vector may comprise a nucleic acid sequence encoding a CRISPR enzyme.
  • the nucleic acid sequence encoding the CRISPR enzyme may be contained in one vector, or the nucleic acid sequence encoding the CRISPR enzyme may be divided into several vectors.
  • the vector may comprise one or more control / control components.
  • the regulatory / control component may include a capture motor, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor, and / or a 2A Sequence.
  • a capture motor an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor, and / or a 2A Sequence.
  • the promoter may be a promoter recognized by RNA polymerase II.
  • the promoter may be a promoter recognized by RNA polymerase III.
  • the promoter may be an inducible promoter.
  • the promoter may be a target specific promoter.
  • the promoter may be a virus or a non-viral promoter.
  • the promoter may be a suitable promoter depending on the regulatory region (i. E., The nucleic acid sequence encoding the gRNA and / or CRISPR enzyme).
  • a promoter useful for gRNA may be a H1, EF-1a, tRNA or U6 promoter.
  • promoters useful for CRISPR enzymes may be CMV, EF-1a, EFS, MSCV, PGK or CAG promoters.
  • the vector may be a viral vector or a recombinant viral vector.
  • the virus may be a DNA virus or an RNA virus.
  • the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.
  • dsDNA double-stranded DNA
  • ssDNA single-stranded DNA
  • the RNA virus may be a single stranded RNA (ssRNA) virus.
  • ssRNA single stranded RNA
  • the virus may be, but is not limited to, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus or herpes simplex virus.
  • the nucleic acid sequence encoding the gRNA and / or CRISPR enzyme may be delivered or introduced by a recombinant lentivirus.
  • nucleic acid sequences encoding gRNA and / or CRISPR enzymes can be delivered or introduced by recombinant adenoviruses.
  • nucleic acid sequences encoding gRNA and / or CRISPR enzymes can be delivered or introduced by recombinant AAV.
  • nucleic acid sequence encoding the gRNA and / or CRISPR enzyme may be delivered or introduced by a hybrid of at least one of a hybrid virus, e. G., A virus as described herein.
  • a hybrid virus e. G., A virus as described herein.
  • it may be delivered or introduced into a subject in the form of a gRNA-CRISPR enzyme complex.
  • the gRNA may be DNA, RNA, or a mixture thereof.
  • the CRISPR enzyme may be in the form of a peptide, polypeptide or protein.
  • the gRNA and CRISPR enzymes can be delivered or introduced into the subject in the form of RNA-type gRNA and protein-form CRISPR gRNA-CRISPR enzyme complex, or ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • the CRISPR gRNA-CRISPR enzyme complex is described by electroporation, microinjection, transient cell compaction or squeezing (see, for example, Lee, et al, (2012) Nano Lett., 12, 6322-6327) ), Lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery, or a combination thereof.
  • the gRNA-CRISPR enzyme complexes disclosed by the present invention can be used to artificially manipulate or modify a target gene, that is, a retinal function-forming gene.
  • the target gene can be manipulated or modified using the above-described gRNA-CRISPR enzyme complex, i.e., CRISPR complex.
  • the manipulation or modification of the target gene includes both the steps of i) cutting or damaging the target gene and ii) repairing or repairing the damaged target gene.
  • cleavage or damage of the target gene may be a cleavage or damage of the target gene using the CRISPR complex, specifically, a cleavage of the target sequence in the target gene.
  • the target sequence may be the target of the gRNA-CRISPR enzyme complex, and the target sequence may or may not include the PAM sequence recognized by the CRISPR enzyme. These target sequences can provide the operator with important criteria in the gRNA design stage.
  • the target sequence can be specifically recognized by the gRNA of the gRNA-CRISPR enzyme complex, which allows the gRNA-CRISPR enzyme complex to be located close to the recognized target sequence.
  • Cleavage " of the target site refers to the breakage of the covalent backbone of the polynucleotide. Cleavage can include, but is not limited to, enzymatic or chemical hydrolysis of phosphodiester linkages, and can be accomplished by a variety of other methods. Both single-strand breaks and double-strand breaks are possible, and double-strand breaks can occur as a result of two distinct single-strand breaks. Cleavage of double strands can produce blunt ends or staggered ends (or sticky ends).
  • the cleavage or deletion of a target gene using the CRISPR complex may be such that all of the double strands of the target sequence are cleaved or damaged.
  • the CRISPR complex can cleave both of the double strands of the target sequence that make a complementary binding to the gRNA.
  • each CRISPR complex can cleave two single strands of a target sequence that make a complementary binding to the gRNA, respectively . That is, the complementary single strand of the target sequence that is complementary to the gRNA is cleaved by SpCas9 nicase (D10A), and the non-complementary single strand of the target sequence that is complementary to the gRNA is cleaved by SpCas9 nicase (H840A) And each cutting may occur sequentially or simultaneously.
  • the cleavage or deletion of the target gene or nucleic acid using the CRISPR complex may be such that only a single strand of the double strand of the target sequence is cut or damaged.
  • the single strand may be a guide nucleic acid binding sequence of a target sequence that is complementary to a gRNA, that is, a complementary single strand, or a guide nucleic acid non-binding sequence that does not have a complementary binding with a gRNA, It can be a single strand.
  • the CRISPR complex when the CRISPR enzyme is SpCas9 nicarase (D10A), the CRISPR complex has a guide nucleic acid binding sequence of the target sequence that is complementary to the gRNA, i.e., the complementary single strand is cleaved by SpCas9 nicase (D10A) And does not cleave the guide nucleic acid non-binding sequence of the target sequence that does not make a complementary binding with the gRNA, that is, the gRNA and the non-complementary single strand.
  • the CRISPR complex when the CRISPR enzyme is SpCas9 nicase (H840A), the CRISPR complex does not have a complementary binding with the gRNA, and the guide nucleic acid non-binding sequence of the target sequence, that is, the gRNA and the non- (H840A), and the guide nucleic acid binding sequence of the target sequence that is complementary to the gRNA, i.e., the complementary single strand, may not be cleaved.
  • the guide nucleic acid non-binding sequence of the target sequence that is, the gRNA and the non- (H840A)
  • the guide nucleic acid binding sequence of the target sequence that is complementary to the gRNA i.e., the complementary single strand
  • cleavage or damage of a target gene or nucleic acid using the CRISPR complex may be to remove some nucleic acid fragments.
  • the double strand of the target sequence that makes a complementary binding with the first gRNA is cleaved, 2 < / RTI > gRNA by cleaving the double strand of the target sequence that is complementary to the second gRNA and the second gRNA and SpCas9.

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Abstract

La présente invention concerne une composition de manipulation de gène pour le traitement ou le soulagement d'un trouble de dysfonctionnement rétinien et une méthode l'utilisant. Plus spécifiquement, la présente invention concerne une composition de manipulation de gène comprenant un acide nucléique de guidage capable de cibler un gène de rétinogenèse et une méthode de traitement ou de soulagement de maladies provoquées par un dysfonctionnement rétinien consistant à manipuler et/ou éditer artificiellement un gène de rétinogenèse à l'aide de la composition.
PCT/KR2018/011522 2017-09-29 2018-09-28 Manipulation de gène pour le traitement d'un trouble de dysfonctionnement rétinien Ceased WO2019066549A2 (fr)

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CN111876418A (zh) * 2020-07-17 2020-11-03 陕西希诺谷生物科技有限公司 先天性黑蒙模型犬的建立方法
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CN112522292A (zh) * 2020-10-29 2021-03-19 南京启真基因工程有限公司 一种用于构建先天性黑蒙症克隆猪核供体细胞的CRISPR/Cas9系统及其应用
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